TWI671403B - Method for controlling controlled assembly of polypeptide - Google Patents

Abstract

It was found that the assembly can be inhibited by changing the amino acid residue that forms the interface during the assembly between polypeptides to an amino acid with the same charge. According to the present invention, hetero molecules can be efficiently formed.

The present invention is applicable, for example, when producing a bispecific antibody.

Description

Manufacturing method of controlled assembly polypeptide

The present invention relates to a method for manufacturing a polypeptide controlled by intramolecular or intermolecular assembly, a polypeptide with controlled assembly within or between molecules, and a pharmaceutical composition containing the polypeptide as an active ingredient.

Antibodies have high stability in the blood and have few side effects, so they are valued as pharmaceuticals. One of them is a bispecific antibody that recognizes both antigens. Currently, MDX-210 is a IgG bispecific antibody that is undergoing clinical trials. It is an antibody that retargets mononuclear spheres expressing FcγRI to cancer cells expressing HER-2 / neu (see non-patent literature). 1). Antibody manufacturing generally uses genetic recombination techniques. Specifically, DNA encoding an antibody-producing protein is cloned from antibody-producing cells such as fusion tumors, antibody-sensing lymphocytes, or phage libraries suggesting antibody genes, and is embedded in an appropriate vector. Etc. Introduction of host cell technology for production. The production of IgG type bispecific antibodies using genetic recombination technology is to introduce the genes of the H chain and L chain of the two IgGs of interest into a total of 4 genes and secrete them by co-expression. As shown in this expression, when the wild-type H chain and L chain constitutive genes are expressed, the two covalent bonds of the H chain or the non-covalent bonds of the H chain and the L chain occur randomly, so the purpose is dual-specific The proportion of sexual antibodies becomes extremely low. Specifically, only one of the ten bispecific antibodies has low production efficiency. Production efficiency of target antibody A low rate will not only hinder the purification of the target antibody, but also increase the non-uniformity such as the difference between batches, which will increase the production cost.

From the viewpoint of the efficient production of bispecific antibodies, it has been reported that by replacing the CH3 region of the IgG H chain with an amino acid, preferential secretion of IgG in which the H chain is a heterogeneous combination (see Patent Document 1) And non-patent documents 2 and 3). Specifically, the amino acid side chain existing in the CH3 region of one of the H chains is replaced with a larger side chain (knob; protrusion), and the amino acid side chain existing in the CH3 region of the other H chain is replaced Instead of a small side chain (hole; void), the protrusions are arranged in the void to promote the formation of heterogeneous H chains and inhibit the formation of homogeneous H chains. It has also been reported that the interface between the H-chain variable region (hereinafter referred to as VH) and the L-chain variable region (hereinafter referred to as VL) uses the same "protrusion" and "gap" (see Non-Patent Document 4). . According to the report by Zhe et al., By replacing two types of amino acids present at the interface of VH and VL (four types of two chains), the formation of hetero molecules is promoted 1.28 times more efficiently (wild type; 72%, variants; 92 %). In addition, one amino acid substitution (two types in both chains) has about the same efficiency as the wild type. However, the method of providing knobs and holes in VH and VL cannot be said to sufficiently promote the formation of foreign molecules.

[Patent Document 1] International Publication No. 96/27011

[Non-Patent Document 1] Segal DM et al., Current Opinion in Immunology, 1999, Vol. 11, p. 558-562

[Non-Patent Document 2] Ridgway JB et al., Protein Engineering, 1996, Vol. 9, p. 617-621

[Non-Patent Document 3] Merchant AM, Nature Biotechnology, 1998, Vol. 16, p. 677-681

[Non-Patent Document 4] Zhe Z et al., Protein Science, 1997, Vol. 6, p. 781-788

The present invention was conceived in view of this situation, and its purpose is to provide a method for controlling the assembly of a polypeptide, an assembly of a controlled polypeptide, and a method for manufacturing the polypeptide. Another aspect of the present invention is to provide a method for efficiently manufacturing a bispecific antibody by controlling the assembly of the interface between VH and VL. Another object is to provide a method for efficiently producing one structural isomer of sc (Fv) 2.

The present inventors have selected VH and VL of antibodies for polypeptides for assembly control, and have worked hard on methods to control the assembly of these VH and VL.

As a result, it was found that by replacing the amino acid existing at the interface between VH and VL with a charged amino acid, the assembly of VH and VL can be suppressed, which is more efficient than the method using the above-mentioned protrusions and voids. Formation of hetero molecules.

Surprisingly, according to the method of the present invention, it is possible to efficiently form a hetero molecule only by substituting one amino acid (two amino acids in total, VH and VL) existing at the interface between VH and VL. Yes, from the viewpoint of antigenicity, the less the amino acid substitution, the better. According to one aspect of the present invention, only one amino acid existing at the interface between VH and VL can effectively form a hetero molecule.

That is, VH can be controlled by the findings discovered by the present inventors, etc. Assembly with VL. In addition, the present invention can be applied not only to the control of assembly of VH and VL, but also to the control of assembly between arbitrary polypeptides.

In addition, the present inventors confirmed that the bispecific antibody obtained by the assembly control method of the present invention maintains its function.

As described above, the present inventors have successfully developed a method capable of controlling assembly between arbitrary polypeptides, and completed the present invention.

The present invention relates to a method for controlling the assembly of a polypeptide, a polypeptide to be controlled for assembly, and a method for manufacturing the polypeptide. More specifically, it provides the following:

[1] A method for manufacturing a polypeptide variant, which comprises mutating the amino acid residues forming the internal interface of the polypeptide to control the assembly of the polypeptide, comprising the following steps: (a) encoding the amino acid residues forming the internal interface of the polypeptide The nucleic acid is altered from the original nucleic acid in a manner that inhibits assembly within the polypeptide, (b) culturing the host cell to express the nucleic acid, and (c) recovering the polypeptide from the host cell culture.

[2] A method for producing heteromultimers, which comprises mutating the amino acid residues forming the interface between polypeptides to control the assembly of heteromultimers, including the following steps: (a) encoding the amines that form the interfaces between polypeptides The nucleic acid of the amino acid residue is changed from the original nucleic acid in such a manner that the assembly between the polypeptides is inhibited, (b) the host cell is cultured to express the nucleic acid, and (c) the heteromultimer is recovered from the host cell culture.

[3] The method according to [1], wherein, among the polypeptides capable of forming two or more structural structural isomers, a nucleic acid encoding an amino acid residue forming an internal interface of the polypeptide is formed so as to form one or more structures The manner in which the assembly of the structural isomer polypeptide is inhibited is altered from the original nucleic acid.

[4] The method according to [2], wherein, among heteromultimers capable of forming two or more kinds of multimers, a nucleic acid encoding an amino acid residue forming an interface between polypeptides is subjected to The manner in which the assembly of polypeptides forming more than one multimer is inhibited is altered from the original nucleic acid.

[5] The method according to [1] or [2], wherein the change of step (a) is to change the original nucleic acid to a method in which the amino acid residues of two or more residues forming the interface have the same charge. Variations of amino acid residues introduced at the interface.

[6] The method according to [5], wherein the amino acid residue introduced is glutamic acid (E).

[7] The method according to [5], wherein the introduced amino acid residue is aspartic acid (D).

[8] The method according to [5], wherein the introduced amino acid residue is lysine (K).

[9] The method according to [5], wherein the amino acid residue introduced is arginine (R).

[10] The method according to [5], wherein the amino acid residue introduced is histamine (H).

[11] The method according to [1] or [2], wherein the change in step (a) is to change the original nucleic acid so that the amino acid residue forming the hydrophobic core at the interface becomes a charged amino group The method of acid residue introduces a variation of amino acid residue at this interface.

[12] The method according to [11], wherein the introduced amino acid residue is glutamic acid (E).

[13] The method according to [11], wherein the introduced amino acid residue is aspartic acid (D).

[14] The method according to [11], wherein the amino acid residue introduced is lysine (K).

[15] The method according to [11], wherein the introduced amino acid residue is arginine (R).

[16] The method according to [11], wherein the amino acid residue introduced is histidine (H).

[17] The method according to [1] or [2], wherein the interface of the polypeptide is formed by the variable region of the heavy chain and the variable region of the light chain of the antibody.

[18] The method according to [1] or [2], wherein the interface of the polypeptide is formed by two or more heavy chain variable regions.

[19] The method of [1] or [2], wherein the interface of the polypeptide is formed by the heavy chain constant region and the light chain constant region of the antibody.

[20] The method according to [1] or [2], wherein the interface of the polypeptide is formed by two or more heavy chain invariant regions.

[21] The method according to [1], wherein the polypeptide is a single-chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are bound by a linker.

[22] The method of [2], wherein the heteromultimer system includes a multispecific antibody having two or more types of variable regions of a heavy chain and two or more types of variable regions of a light chain.

[23] The method according to [22], wherein the heteromultimer is a bispecific antibody.

[24] A polypeptide variant or heteromultimer is produced by the method of [1] or [2].

[25] A polypeptide variant that changes the amino acid residues that form the interface within the polypeptide to inhibit assembly in the original polypeptide.

[26] A heteromultimer that changes the amino acid residues that form the interface between the polypeptides to inhibit the assembly between the original polypeptides.

[27] The polypeptide variant of [25], wherein the original polypeptide can form two or more kinds of structural isomers.

[28] The heteromultimer according to [26], wherein the original polypeptide can form two or more kinds of multimers.

[29] The polypeptide variant of [25] or the heteromultimer of [26], wherein the change of the amino acid residue forming the interface of the aforementioned polypeptide is a variation of the amino acid residue introduced at the interface to form Amino acid residues with more than 2 residues at the interface become the same charge.

[30] The polypeptide variant or heteromultimer according to [29], wherein the amino acid residue introduced is glutamic acid (E).

[31] The polypeptide variant or heteromultimer according to [29], wherein the amino acid residue introduced is aspartic acid (D).

[32] The polypeptide variant or heteromultimer according to [29], wherein the amino acid residue introduced is lysine (K).

[33] The polypeptide variant or heteromultimer according to [29], wherein the amino acid residue introduced is arginine (R).

[34] The polypeptide variant or heteromultimer according to [29], wherein the amino acid residue introduced is histidine (H).

[35] The polypeptide variant of [25] or the heteromultimer of [26], wherein the change of the amino acid residue forming the interface of the aforementioned polypeptide is a variation of the amino acid residue introduced at the interface to form The amino acid residues present in the hydrophobic core of the interface have a charge.

[36] The polypeptide variant or heteromultimer according to [35], wherein the amino acid residue introduced is glutamic acid (E).

[37] The polypeptide variant or heteromultimer according to [35], wherein the amino acid residue introduced is aspartic acid (D).

[38] The polypeptide variant or heteromultimer according to [35], wherein the amino acid residue introduced is lysine (K).

[39] The polypeptide variant or heteromultimer according to [35], wherein the amino acid residue introduced is arginine (R).

[40] The polypeptide variant or heteromultimer according to [35], wherein the amino acid residue introduced is histidine (H).

[41] The polypeptide variant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by a variable region of a heavy chain and a variable region of a light chain of an antibody.

[42] The polypeptide variant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by two or more kinds of variable regions of heavy chains.

[43] The polypeptide variant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by the heavy chain invariant region and the light chain invariant region of the antibody.

[44] The polypeptide variant of [25] or the heteromultimer of [26], wherein the polypeptide interface is formed by two or more heavy chain invariant regions.

[45] The polypeptide variant of [25], wherein the polypeptide is a single-chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are linked by a linker.

[46] The heteromultimer according to [26], wherein the heteromultimer system includes a multispecific antibody having two or more heavy chain variable regions and two or more light chain variable regions.

[47] The heteromultimer according to [46], wherein the heteromultimer is a bispecific antibody.

[48] A composition comprising the polypeptide variant of [25] or the heteromultimer of [26], and a pharmaceutically acceptable carrier.

[49] A nucleic acid encoding the polypeptide variant of [25] or a heteromultimer of [26].

[50] A host cell having the nucleic acid of [49].

[51] A method for producing a polypeptide variant of [25] or a heteromultimer of [26], comprising the steps of: culturing the host cell of [50], and recovering the polypeptide from the cell culture.

[52] A method for controlling assembly of a polypeptide, comprising changing an amino acid residue that forms an interface within the original polypeptide to inhibit assembly within the polypeptide.

[53] A method for controlling the assembly of heteromultimers, including The amino acid residues at the interface between the peptides are changed to inhibit assembly between the polypeptides.

[54] The method according to [52], wherein, among the polypeptides capable of forming two or more structural isomers, the amino acid residues forming the internal interface of the polypeptide are changed to suppress the formation of one or more structural isoforms. Assembly of polypeptides of the structure.

[55] The method according to [53], wherein among the heteromultimers capable of forming more than two kinds of multimers, the amino acid residues forming the interface between the polypeptides are changed to inhibit the formation of more than one kind of multimers Assembly between peptides in the body.

[56] The method according to [52] or [53], wherein the change of the amino acid residue forming the interface of the aforementioned polypeptide is a variation of an amino acid residue introduced at the interface so that two residues forming the interface are formed The above amino acid residues become charged with the same kind.

[57] The method according to [56], wherein the introduced amino acid residue is glutamic acid (E).

[58] The method according to [56], wherein the introduced amino acid residue is aspartic acid (D).

[59] The method according to [56], wherein the introduced amino acid residue is lysine (K).

[60] The method according to [56], wherein the introduced amino acid residue is arginine (R).

[61] The method according to [56], wherein the amino acid residue introduced is histidine (H).

[62] The method according to [52] or [53], wherein the change of the amino acid residue forming the interface of the aforementioned polypeptide is a variation of the amino acid residue introduced at the interface so that a hydrophobic core existing at the interface is formed The amino acid residue becomes a charged amino acid residue.

[63] The method according to [62], wherein the introduced amino acid residue is glutamic acid (E).

[64] The method according to [62], wherein the introduced amino acid residue is aspartic acid (D).

[65] The method according to [62], wherein the amino acid residue introduced is lysine (K).

[66] The method according to [62], wherein the amino acid residue introduced is arginine (R).

[67] The method according to [62], wherein the introduced amino acid residue is histidine (H).

[68] The method according to [52] or [53], wherein the polypeptide interface is formed by a heavy chain variable region and a light chain variable region of the antibody.

[69] The method according to [52] or [53], wherein the polypeptide interface is formed by two or more kinds of heavy chain variable regions.

[70] The method according to [52] or [53], wherein the polypeptide interface is formed by a heavy chain constant region and a light chain constant region of the antibody.

[71] The method according to [52] or [53], wherein the polypeptide interface is formed of two or more heavy chain invariant regions.

[72] The method according to [52], wherein the polypeptide is a single-chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are bound by a linker.

[73] The method according to [53], wherein the heteromultimer system includes a multispecific antibody having two or more heavy chain variable regions and two or more light chain variable regions.

[74] The method of [73], wherein the heteromultimer is a bispecific antibody.

[75] An antibody comprising a heavy chain variable region and a light chain variable region. The following (1) and (2) amino acid residues are amino acid residues having the same charge: (1) contained in the heavy chain The amino acid residue in the variable region is equivalent to position 39 (glutamine) in the amino acid sequence of SEQ ID NO: 6; (2) the amino acid residue included in the variable region of the light chain is equivalent to SEQ ID NO: 44 of the amino acid sequence of 8 (glutamine).

[76] An antibody comprising a heavy chain variable region and a light chain variable region. The following amino acid residues (1) and (2) are amino acid residues having the same charge: (1) included in the heavy The amino acid residue in the variable region of the chain is equivalent to the 45th position (leucine) in the amino acid sequence of sequence number: 6; (2) The amino acid residue contained in the variable region of the light chain corresponds to position 50 (proline) in the amino acid sequence of SEQ ID NO: 8.

[77] An antibody comprising a heavy chain variable region and a light chain variable region, and any one of (1) or (2) below is a charged amino acid residue: (1) included in the heavy chain variable region The amino acid residue corresponds to position 45 (leucine) in the amino acid sequence of SEQ ID NO: 6; (2) the amino acid residue contained in the variable region of the light chain corresponds to the sequence number: Position 50 in the amino acid sequence of 8 (proline).

[78] The antibody of [75] or [76], wherein the aforementioned amino acid residues having the same charge are selected from amino acid residues contained in any of the following groups (a) or (b): (a ) Glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), and histidine (H).

[79] The antibody of [77], wherein the charged amino acid residues are glutamic acid (E), aspartic acid (D), lysine (K), and spermine (R ) Or Histidine (H).

[80] The antibody according to any one of [75] to [77], wherein the polypeptide is a single-chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are bound by a linker.

[81] The antibody according to any one of [75] to [77], wherein the polypeptide is a multispecific antibody comprising two or more heavy chain variable regions and two or more light chain variable regions.

[82] The antibody of [81], wherein the polypeptide is a bispecific antibody.

[83] A composition comprising the antibody of any one of [75] to [77] and a pharmaceutically acceptable carrier.

[84] A nucleic acid encoded as many antibodies constituting any one of [75] to [77] Peptide.

[85] A host cell having the nucleic acid of [84].

[86] A method for producing an antibody according to any one of [75] to [77], comprising the steps of: culturing the host cell of [85], and recovering the polypeptide from the cell culture.

[87] An antibody comprising two or more heavy chain CH3 regions. Among the first heavy chain CH3 regions, one of the groups consisting of the amino acid residues shown in (1) to (3) below is selected from one to three. Histidine residues have the same charge.

(1) Amino acid residues with EU numbers 356 and 439 are included in the CH3 region of the heavy chain.

(2) Amino acid residues with EU numbers 357 and 370 are included in the CH3 region of the heavy chain.

(3) Amino acid residues with EU numbers 399 and 409, including the heavy chain CH3 region.

[88] The antibody according to [87], which is an amino acid residue group selected from the group of amino acid residues shown in (1) to (3) above in the second heavy chain CH3 region, corresponding to the aforementioned first In the heavy chain CH3 region, the amino acid residues of groups 1 to 3 shown in the above (1) to (3) with the same kind of charge are carried with the same charges as those in the first heavy chain CH3 region. The corresponding amino acid residue in the charge.

[89] The antibody of [87], wherein the amino acid residue having the same charge is selected from the amino acid residues contained in any of the following groups (a) or (b): (a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), histidine (H),

[90] the antibody of [87], wherein the first heavy chain CH3 region and the second heavy chain The CH3 region of the heavy chain is crosslinked with a disulfide bond.

[91] The antibody according to [87], which has two or more heavy chain invariant regions.

[92] The antibody of [87] is a multispecific antibody comprising two or more heavy chain variable regions and two or more light chain variable regions.

[93] The antibody of [92] is a bispecific antibody.

[94] A composition comprising the antibody of [87] and a pharmaceutically acceptable carrier.

[95] A nucleic acid encoding a polypeptide constituting the antibody of [87].

[96] A host cell having the nucleic acid of [95].

[97] A method for producing an antibody, the antibody of [87], comprising the steps of: culturing the host cell of [96], and recovering the polypeptide from the cell culture.

Figure 1 shows the pattern of the Fv region of humanized SB04. (A) shows amino acid residues H39 and L38 at the interface of VH and VL, and (B) amino acid residues H45 and L44 at the interface of VH and VL. .

Figure 2 shows the evaluation results of H chain L chain assembly in H39 and L38 altered antibodies. As a result, the comparison of all modified antibodies with the wild type showed that the assembly ratio of the antibody of interest increased.

Description of the line

M: molecular weight marker

1: Humanized XB12 H chain (Q) + Humanized XB12 L chain (Q)

2: Humanized XB12 H chain (Q) + Humanized SB04 L chain (Q)

3: Wild type: Humanized XB12 H chain (Q) + Humanized XB12 L chain (Q)

+ Humanized SB04 L chain (Q)

4: D variant: humanized XB12 H chain (D) + humanized XB12 L chain (Q) + humanized SB04 L chain (D)

5: E variant: humanized XB12 H chain (E) + humanized XB12 L chain (Q) + humanized SB04 L chain (E)

6: R variant: humanized XB12 H chain (R) + humanized XB12 L chain (Q) + humanized SB04 L chain (R)

7: K variant: humanized XB12 H chain (K) + humanized XB12 L chain (Q) + humanized SB04 L chain (K)

Figure 3 shows the results of evaluation of the coagulation activity among the modified antibodies of H39 and L38. As a result, the bispecific antibodies in which the XB12 H chain (H39) and SB04 L chain (L38) were changed to Glu showed coagulation activity equal to or more than that of the wild type.

Figure 4 shows the results of evaluation of FactorIXa binding activity among the modified antibodies of H39 and L38. As a result, among all the altered antibodies, the binding activity was equivalent to that of the wild type.

Figure 5 shows the evaluation results of FactorX-binding activity among the modified antibodies of H39 and L38. As a result, among all the modified antibodies, the binding activity was equivalent to that of the wild type.

Fig. 6 shows the evaluation results of the assembly of H chain and L chain among the modified antibodies of L44. As a result, compared with the wild type, the assembly ratio of the target antibody increased among all the changed antibodies.

Description of the line

1: Wild type: humanized XB12 H chain + humanized XB12 L chain (P) + humanized SB04 L chain (P)

2: D variant: humanized XB12 H chain + humanized XB12 L chain (P) + humanized SB04 L chain (D)

3: E variant: humanized XB12 H chain + humanized XB12 L chain (P) + humanized SB04 L chain (E)

4: R variant: humanized XB12 H chain + humanized XB12 L chain (P) + humanized SB04 L chain (R)

5: K variant: humanized XB12 H chain + humanized XB12 L chain (P) + humanized SB04 L chain (K)

Fig. 7 shows the results of evaluation of coagulation activity among L44-modified antibodies. As a result, among all the modified antibodies, it showed higher coagulation activity than the wild type.

Figure 8 shows the evaluation results of FactorX-binding activity among L44-modified antibodies. As a result, among all the modified antibodies, the binding activity was equivalent to that of the wild type.

Figure 9 shows the evaluation results of the assembly of H chain and L chain among the modified antibodies of H39, L38 and L44. As a result, compared with the wild type, among all the changed antibodies, it was shown that the assembly ratio of the antibody of interest increased.

Description of the line

1: Wild type: Humanized XB12 H chain (H39: Q) + Humanized XB12 L chain (L38: Q) + Humanized SB04 L chain (L38: Q, L44: P)

2: E + D variant: humanized XB12 H chain (H39: E) + humanized XB12 L chain (L38: Q) + humanized SB04 L chain (L38: E, L44: D)

3: E + E variant: humanized XB12 H chain (H39: E) + humanized XB12 L chain (L38: Q) + humanized SB04 L chain (L38: E, L44: E)

4: E + R variant: humanized XB12 H chain (H39: E) + humanized XB12 L chain (L38: Q) + humanized SB04 L chain (L38: E, L44: R)

5: E + K variant: humanized XB12 H chain (H39: E) + humanized XB12L chain (L38: Q) + humanized SB04 L chain (L38: E, L44: K)

M: molecular weight marker

Figure 10 shows the results of evaluation of coagulation activity among the modified antibodies of H39, L38, and L44. As a result, the XB12 H chain (H39) and SB04 L chain (L38, L44) altered bispecific antibodies showed coagulation activity equal to or more than that of the wild type.

Fig. 11 shows the evaluation results of FactorIXa binding activity among the modified antibodies of H39, L38 and L44. As a result, among all the modified antibodies, the binding activity was equivalent to that of the wild type.

FIG. 12 is a schematic diagram showing an example of an sc (Fv) 2 structure including two types of heavy chain variable regions (VH1 and VH2) and two types of light chain variable regions (VL1 and VL2). (a) Structural sc (Fv) 2 mainly exists in two types of structural isomers as shown in (b).

Figure 13 shows the results of the structural isomers of u2-wz4, peak1 and peak2, separated by cation exchange chromatography.

Figure 14 shows the peptide map of peak1 and peak2 separated by cation exchange chromatography.

Figure 15 shows the reduced SDS-PAGE results of the structural isomers of u2-wz4, peak1 and peak2, and the separation of u2-wz4 after subtilisin treatment. The resulting band structure is shown on the right.

FIG. 16 shows that the decomposition pattern of the subtilisin-limited decomposition due to the structural difference between the bivalent scFv and the single-chain antibody is different. In the case of a bivalent scFv structure, a low molecular weight fragment surrounded by a dotted line is generated.

Figure 17 shows the structural isomers of u2-wz4, peak1, peak2, and separation. Former u2-wz4 gel filtration chromatography results after limited decomposition with Subtilisin. The arrow indicates the dissolution position of the low molecular weight peak.

Figure 18 shows the gel filtration chromatography results of u2-wz4, variant v1, and variant v3 after purification of the MG10-GST fusion protein immobilized column.

Figure 19 shows the results of cation exchange chromatography with u2-wz4, variant v1, and variant v3.

Figure 20 shows isoelectric point electrophoresis results of u2-wz4, u2-wz4 refined peak1, u2-wz4 refined peak2, variant v1, and variant v3.

Figure 21 shows the results of gel filtration chromatography analysis of the protease-limited decomposition of u2-wz4 refined peak1, u2-wz4 refined peak2, variant v1, and variant v3.

Fig. 22 shows the results of evaluation of TPO-like synergistic activity of the purified peaks of u2-wz4, peak2 of u2-wz4, variant v1, and variant v3.

Figure 23 shows the results of DSC analysis of u2-wz4 refined peak1, u2-wz4 refined peak2, variant v1, and variant v3.

FIG. 24 shows the monomer residual rate obtained by gel filtration chromatography analysis in the thermal accelerated test of u2-wz4 refined peak1, u2-wz4 refined peak2, variant v1, and variant v3.

Figure 25 shows the proportion of structural isomers obtained by cation exchange chromatography analysis in the thermal accelerated test of u2-wz4 refined peak1, u2-wz4 refined peak2, variant v1, and variant v3.

Figure 26 shows the results of the evaluation of the coagulation activity of humanized bispecific antibodies (Humanized A69 (hA69-PFL) / Humanized B26 (hB26-PF) / Humanized BBA (hAL-AQ)). The results showed that the coagulation activity was more than or equal to that of the chimeric bispecific antibody. Sex.

FIG. 27 is a conceptual diagram showing that the formation efficiency of a bispecific antibody is improved by changing the invariant region of the H chain. The EU number (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH) was used to change the position number.

Figure 28 shows the IEX analysis chromatogram of a humanized bispecific antibody (IgG4 type) with a modified CH3 interface.

FIG. 29 shows the formation ratios of A-Homo, BiAb, and B-Homo obtained by IEX analysis of a humanized bispecific antibody (IgG4) with a changed CH3 interface.

Fig. 30 shows the monomer residual rate of the BiAb purified from a humanized bispecific antibody (IgG4) with a changed CH3 interface after a thermal acceleration test at 60 ° C-1W.

Figure 31 shows the results of the coagulation activity evaluation of a humanized bispecific antibody (IgG4 type) with a modified CH3 interface. The results showed the same coagulation activity as the unchanged bispecific antibody.

Figure 32 shows the formation ratio of the humanized bispecific antibody (IgG1 type) with the CH3 interface changed by A-Homo, BiAb, and B-Homo analysis by IEX.

[Best Mode for Implementing Invention]

The present invention relates to a method for controlling the assembly of polypeptides or the assembly of heteromultimers composed of polypeptides.

First, the present invention provides a method for controlling the assembly of a polypeptide, which comprises changing the amino acid residues forming the internal interface of the original polypeptide to inhibit the internal organization of the polypeptide. Installed.

Polypeptides in the present invention generally refer to peptides and proteins with a length of about 10 amino acids or more. The peptide is usually derived from a biological organism, but is not particularly limited. For example, it may be a polypeptide composed of artificially designed sequences. In addition, it may be any of a natural polypeptide, a synthetic polypeptide, and a recombinant polypeptide. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

The assembly of the polypeptide in the present invention, in other words, can refer to a state where, for example, two or more polypeptide regions interact.

In the present invention, "controlling assembly" means controlling the desired assembly state, and more specifically, controlling so that undesired assembly is not formed in the polypeptide.

In the present invention, "interface" refers to the assembly surface during normal assembly (interaction), and the amino acid residue forming the interface refers to the 1 or most amino acid residues contained in the polypeptide region usually used for assembly, more Preferred are amino acid residues that are close to and associated with the interaction during assembly. Specifically, the interaction includes a case where amino residues which are close to each other during assembly form hydrogen bonds with each other, an electrostatic interaction, a case of a salt bridge, and the like.

In the present invention, the "amino acid residue forming an interface", in more detail, refers to the amino acid residue contained in the polypeptide region constituting the interface. The polypeptide region constituting the interface, if taken as an example, refers to a polypeptide region in an antibody, a ligand, a receptor, a matrix, and the like, which is responsible for selective binding within or between molecules. Specific examples include heavy chain variable regions and light chain variable regions among antibodies.

In the method of the present invention, the "change" of the amino acid residue specifically refers to replacing the original amino acid residue with another amino acid residue to make the original amine The deletion of an amino acid residue, the addition of a new amino acid residue, and the like preferably refer to the replacement of an ortho amino acid residue with another amino acid residue.

The "polypeptide" in the present invention is preferably a polypeptide capable of forming two or more structural isomers. Generally speaking, the structural structural isomers refer to proteins having the same amino acid sequence but different three-dimensional structures (cube structures) from each other. Generally, at least one of the chemical or physical properties of the structural isomers differs from each other.

A preferred aspect of the present invention relates to a method for preferentially (efficiently) obtaining a desired structural structural isomer from among two or more structural structural isomers. That is, in one aspect, it is related to changing the amino acid residues forming the internal interface of the polypeptide among polypeptides capable of forming two or more structural isomers to inhibit the formation of one or more structural structures. Method for assembling isomeric polypeptides.

For example, in the case where there are first to fourth polypeptide regions and these two arbitrary regions can be assembled, (1) the first and second polypeptide regions can be assembled, and the third and the third polypeptide regions can be assembled. Assembly of four polypeptide regions, (2) assembly of first and third polypeptide regions, further, assembly of second and fourth polypeptide regions, (3) assembly of first and fourth polypeptide regions, further, second In the case of the third polypeptide region assembly, there are mainly three structural and structural isomers.

In order to obtain preferentially the polypeptides (structural and structural isomers) assembled in the manner described in (1) above, for example, amino acid residues existing at the interface forming the first, third, or tetrapeptide regions may be used. Changes are made to inhibit the assembly of the first and third and fourth polypeptide regions.

The method of the present invention relates to the assembly control of heteromultimers. The method comprises changing the amino acid residues forming the interface between the original polypeptides to inhibit the assembly between the polypeptides.

The “heteromultimer” in the present invention refers to a protein multimer composed of a plurality of polypeptides and the polypeptides can be assembled with each other. In more detail, a "heteromeric polymer" has at least a first polypeptide and a second polypeptide, and the second polypeptide is at least one amino acid residue from the first polypeptide in the amino acid sequence. Different molecules. Although not particularly limited, it is preferred that the heteromultimer has binding specificity for at least two different ligands, antigens, receptors, or matrices. In addition to the "heterodimer" formed by the first and second polypeptides of the heteromultimer, other types of polypeptides may exist. That is, the "heteromeric multimer" of the present invention is not limited to heterodimer, and includes, for example, heterotrimer and heterotetramer.

In a preferred aspect of the above method, for heteromultimers that can form two or more multimers, the amino acid residues that form the interface between the polypeptides are changed to inhibit the formation of one or more multimers. Method of assembly.

For example, in a protein multimer consisting of the first to fourth polypeptides, any two of these polypeptides can be assembled, mainly including: (1) the first and second polypeptides are assembled, and , Multimers of the third and fourth polypeptide assemblies, (2) first and third polypeptide assemblies, and (2) multimers of the second and fourth polypeptide assemblies, or (3) first and third polypeptides. Four polypeptide assemblies, and multimers of the second and third polypeptide assemblies.

In order to preferentially obtain the situation of multimers assembled in the manner described in (1) above, for example, the amino acid residues contained in the first, three, or tetrapeptides can be changed to inhibit the first poly The peptide is assembled with the third and fourth polypeptides.

Preferred aspects and characteristics of the assembly control method of the polypeptide of the present invention For example, the change of the amino acid residue forming the interface of the polypeptide is a variation of introducing an amino acid residue into the interface, so that the amino acid residue having more than 2 residues forming the interface becomes the same kind of charge.

In the above method, it is considered that the amino acid residues are assembled with each other by changing two or more amino acid residues related to assembly in the interface to have the same charge with each other, and by the repulsive force of the charges. Suppressed.

Therefore, in the above method, the amino acid residues to be changed are preferably two or more amino acid residues which are close to each other during assembly between the polypeptide regions forming the interface.

The amino acid residues that are close during assembly can be found, for example, by analyzing the three-dimensional structure of the polypeptide and examining the amino acid sequence of the polypeptide region that forms the interface during the assembly of the polypeptide. The amino acid residues which are close to each other in the interface become the better target of "change" in the method of the present invention.

Among the amino acids, charged amino acids are known. In general, positively charged amino acids (positively charged amino acids) are known as lysine (K), arginine (R), and histidine (H). As the negatively charged amino acid (negatively charged amino acid), aspartic acid (D), glutamic acid (E), and the like are known. Therefore, preferably, the amino acids having the same charge in the present invention refer to each amino acid having a positive charge, or each amino acid having a negative charge.

In the method of the present invention, it is preferable that all amino acid residues that are mutated are changed in such a manner that they have the same charge. It is preferably not limited to this case. For example, the amino acid residues introduced due to the change are the majority In the case of these amino acid residues, a small number of uncharged amino acid residues may also be included.

The number of amino acid residues to be changed in the method of the present invention is not particularly limited For example, in the case of changing the variable region of an antibody, in order not to reduce the binding activity with the antigen and to not increase the antigenicity, it is preferable to make the number of amino acid residues to be changed as small as possible. As shown in the examples described later, the method of the present invention can control assembly by changing two or one of the two amino acid residues close to each other at the interface. The above-mentioned "minority" refers to, for example, a number of about 1 to 10, preferably a number of about 1 to 5, more preferably a number of about 1 to 3, and most preferably 1 or 2.

In a preferred aspect of the present invention, the amino acid residues introduced (provided to be changed) by the change are preferably all the amino acid residues selected from the above-mentioned positively charged amino acids, or all are selected. Amino acid residues from the negatively charged amino acids described above.

The amino acid residues introduced in the present invention are preferably glutamic acid (E), asparagine (D), lysine (K), arginine (R), and histidine ( H).

In the original (before change) polypeptide, when the amino acid residue (X) forming the interface is already charged, the amino acid residue that is close to and opposite to the amino acid residue during assembly is changed to The amino acid residue (or amino acid residue having the same charge) which is the same as the amino acid residue (X) is also one of the preferred aspects of the present invention. In this aspect, one of the amino acid residues forming the interface may be changed.

In addition, in a preferred aspect of the assembly control method of the present invention, it is characterized in that the change of the amino acid residue forming the peptide interface is a variation of the amino acid residue introduced at the interface, so that the interface is hydrophobic Sexual core amino acid residues become charged amino acid residues.

Generally speaking, a "hydrophobic core" refers to a portion formed by gathering side chains of a hydrophobic amino acid on the inside of an assembled polypeptide. Hydrophobic amino acids include, for example, alanine, isoleucine, leucine, methionine, Phenylalanine, Proline, Tryptophan, Valine, etc. Moreover, the formation of a hydrophobic core may be related to an amino acid residue (for example, tyrosine) other than a hydrophobic amino acid. The hydrophobic core, together with the hydrophilic surface on which the side chain of the hydrophilic amino acid is exposed on the outside, becomes a driving force for promoting the assembly of the water-soluble polypeptide. If the hydrophobic amino acids of the two different functional regions are present on the molecular surface and exposed to water molecules, the entropy increases and the free energy increases. Therefore, in order to reduce and stabilize the free energy, the two functional regions are assembled with each other, and the hydrophobic amino acid at the interface is embedded in the molecule to form a hydrophobic core.

It is considered that when the assembly of the polypeptide occurs, the formation of the hydrophobic core is inhibited because the hydrophobic amino acid that has formed the hydrophobic core is changed to a charged polar amino acid. As a result, the assembly of the polypeptide is inhibited.

For those skilled in the art, by analyzing the amino acid sequence of the desired polypeptide, it is possible to understand whether a hydrophobic core is present and the formation site (region). That is, the present invention is an assembly control method, which is characterized by changing an amino acid residue that can form a hydrophobic core in the interface to a charged amino acid residue.

Among the above methods, in terms of the charged amino acid residues, for example, glutamic acid (E), aspartic acid (D), lysine (K), and arginine (R) are preferred. , Histidine (H).

The assembly control method of the present invention can be used to preferentially obtain (manufacture) an antibody (polypeptide) of interest in the production of an antibody, an antibody fragment, or a polypeptide having similar antibody activity.

In the present invention, the term "antibody" is used in the broadest sense as long as it exhibits the desired biological activity, and includes single antibodies, multiple antibodies, and antibody variants (chimeric antibodies, humanized antibodies, low antibodies). Molecular antibodies (also include Contains antibody fragments), multispecific antibodies, etc.). The "antibody" in the present invention may be any one of a polypeptide and a heteromultimer. Preferred antibodies are monoclonal antibodies, chimeric antibodies, humanized antibodies, and low-molecular-weight antibodies such as antibody fragments. In the present invention, when obtaining (making) such antibodies, the assembly control method of the present invention can be appropriately used.

In the present invention, "multispecific antibodies" (in this specification, the same meaning as "multiple specific antibodies") refer to antibodies that can specifically bind to different multiple epitopes. In other words, the multiple specificity antibody system has specificity for at least two different epitopes. In addition to antibodies that recognize different antigens, it also includes antibodies that recognize different epitopes on the same antigen. (For example, when the antigen is a heteroreceptor, the multispecific antibody recognizes the different functional regions constituting the heteroreceptor, or when the antigen is a monomer, the multispecific antibody recognizes most positions of the monomer antigen). Generally, if this molecular line is bound to two antigens (bispecific antibody: bispecific antibody; in this description, it has the same meaning as "two specific antibodies"), but it can also be used for more species (such as three ) Antigens are specific.

The "antibody" in the present invention includes those whose amino acid sequence is changed by further amino acid substitution, deletion, addition, and / or insertion or chimerization or humanization of the above-mentioned antibody. Amino acid sequence changes by amino acid substitution, deletion, addition and / or insertion, and humanization, chimerization, etc. can be performed by methods known to those skilled in the art. Similarly, in the present invention, the antibody variable region and the constant region used in the production of antibodies by recombinant antibodies can also be substituted, deleted, added, and / or inserted or chimeric or human by amino acid substitution. To change the amino acid sequence.

The antibodies in the present invention may be antibodies derived from various animals, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, camel antibodies, and the like. Further, it may be, for example, a chimeric antibody, and among them, a modified antibody in which an amino acid sequence is substituted, such as a humanized antibody, is preferred. In addition, various antibodies such as antibody modifications, antibody fragments, and low-molecular-weight antibodies that bind various molecules can be used.

A "chimeric antibody" refers to an antibody made by combining sequences derived from different animals. For example, there are antibodies composed of the variable (V) regions of the heavy and light chains of a mouse antibody and the constant (C) regions of the heavy and light chains of a human antibody. The production of chimeric antibodies is well known. For example, chimeric antibodies can be obtained by linking the DNA encoding the V region of the antibody with the DNA encoding the C region of the human antibody, and inserting these into the expression vector and introducing them into the host for production. antibody.

"Humanized antibodies" are also known as reshaped human antibodies, which are antibodies derived from mammals other than humans, such as the complementarity determining region (CDR) of mouse antibodies, and transplanted into the CDRs of human antibodies By. Methods for identifying CDRs are well known (Kabat et al., Sequence of Proteins of Immunological Interest (1987), National Institute of Health, Bethesda, Md .; Chothia et al., Nature (1989) 342: 877). Moreover, its general genetic recombination method is also known (refer to European Patent Application Publication No. EP 125023, WO 96/02576). The CDR of a mouse antibody can be determined by a known method, and DNA encoding an antibody that links the CDR with a framework region (FR) of a human antibody can be obtained, and the humanized antibody can be used as a general expression vector. Department to produce. For example, the DNA can be made by using several oligonucleotides made in such a way that the terminal regions of both CDR and FR have overlapping portions. Primers were synthesized by PCR (see the method of WO98 / 13388). The FR of the human antibody linked via the CDR can be selected by a method in which the CDR forms a good antigen-binding site. If necessary, the amino acid of FR in the variable region of the antibody can also be changed by making the CDRs of the reconstituted human antibody into an appropriate antigen-binding site (Sato et al., Cancer Res. (1993) 53: 851 -6). The amino acid residues in the FR that can be modified include: the portion bound to the antigen by a direct, non-covalent bond (Amit et al., Science (1986) 233: 747-53), the effect on the structure of the CDR or the effect Section (Chothia et al., J. Mol. Biol. (1987) 196: 901-17) and a section related to VH-VL interactions (EP239400 Patent Gazette).

In the present invention, when the antibody is a chimeric antibody or a humanized antibody, the C region of the antibody is preferably derived from a human antibody. For example, Cγ1, Cγ2, Cγ3, and Cγ4 can be used for the H chain, and Cκ and Cλ can be used for the L chain. In order to improve the stability of the antibody or its production, the human antibody C region may be modified as necessary. In the present invention, the chimeric antibody is preferably composed of a variable region derived from a mammal-derived antibody and a constant region derived from a human antibody. On the other hand, the humanized antibody is preferably composed of CDRs derived from mammalian antibodies other than humans and FR and C regions derived from human antibodies. Regarding the variable region, it is organized and explained in (3) -3. The invariant regions derived from human antibodies have inherent amino acid sequences based on IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE equivalent isotypes. In the present invention, the invariant region used by the humanized antibody may be an invariant region of an antibody belonging to any isotype. Preferably, invariant regions of human IgG are used, but are not limited to these. In addition, the FR derived from the human antibody used for the humanized antibody is not particularly limited, and may be an antibody of any isotype.

In the present invention, as long as the variable and invariable regions of the chimeric antibody and the humanized antibody can show the binding specificity of the original antibody, they can also be changed by deletion, substitution, insertion, and / or addition.

Chimeric antibodies and humanized antibodies using human-derived sequences are considered to be useful when administered to humans for therapeutic purposes and the like due to their low antigenicity in humans.

In addition, the low-molecular-weight antibody is useful as an antibody because it can be produced at low cost by using coliforms, plant cells, and the like in terms of its dynamics in vivo.

The antibody fragment is one of low-molecular-weight antibodies. The low-molecular-weight antibody also includes an antibody in which an antibody fragment is part of its structure. In the present invention, as long as the low-molecular-weight antibody has the ability to bind to an antigen, its structure, manufacturing method, and the like are not limited. Among the low-molecular-weight antibodies, antibodies with higher activity than full-length antibodies still exist (Orita et al., Blood (2005) 105: 562-566). In the present specification, the "antibody fragment" is not particularly limited as long as it is a part of a full-length antibody (for example, whole IgG, etc.), but preferably contains a heavy chain variable region (VH) or a light chain variable region ( VL). Examples of preferred antibody fragments include Fab, F (ab ') 2, Fab', and Fv. The amino acid sequence of VH or VL in an antibody fragment can be changed by substitution, deletion, addition and / or insertion. Furthermore, as long as the ability to bind to the antigen is maintained, one of VH and VL may be defective. For example, among the aforementioned antibody fragments, "Fv" is the smallest antibody fragment including a complete antigen recognition site and a binding site. "Fv" is a dimer (VH-VL dimer) strongly bonded by one VH and one VL with a non-covalent bond. Complementarity determining region (CDR) determined by three complementary strands of each variable region An antigen-binding site is formed on the surface of the VH-VL dimer. The six CDRs confer an antigen-binding site on the antibody. However, even if one variable region (or half of the Fv containing only three CDRs specific for the antigen) has a lower affinity than the full binding site, the antigen is still recognized and has the ability to bind. Therefore, molecules smaller than the Fv are also included in the antibody fragments of the present invention. The variable region of the antibody fragment can be chimeric or humanized.

The low-molecular-weight antibody preferably contains both VH and VL. Examples of low-molecular-weight antibodies include antibody fragments such as Fab, Fab ', F (ab') 2, and Fv, and scFv (single-chain Fv) that can be produced using antibody fragments (Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85: 5879-83; Plickthun "The Pharmacology of Monoclonal Antibodies" Vol. 113, edited by Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994)), Diabody (Holliger et al., Proc. Natl. Acad. Sci. USA (1993) 90: 6444-8; EP404097; WO93 / 11161; Johnson et al., Method in Enzymology (1991) 203 : 88-98; Holliger et al., Protein Engineering (1996) 9: 299-305; Perisic et al., Structure (1994) 2: 1217-26; John et al., Protein Engineering (1999) 12 (7) : 597-604; Atwell et al., Mol. Immunol. (1996) 33: 1301-12), sc (Fv) 2 (Hudson et al, J Immunol. Methods (1999) 231: 177-89; Orita et al ., Blood (2005) 105: 562-566), Triabody (Journal of Immunological Methods (1999) 231: 177-89), and Tandem Diabody (Cancer Research (2000) 60: 4336-41) Wait.

Antibody fragments can be obtained by combining antibodies with enzymes such as papaya enzymes, Pepsin and other proteases (see Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al., Science (1985) 229: 81). Furthermore, it can be produced by genetic recombination based on the amino acid sequence of the antibody fragment.

A low-molecular-weight antibody having a structure that changes an antibody fragment can be constructed by obtaining an antibody fragment by enzyme treatment or genetic recombination. Alternatively, genes encoding the entire low-molecular-weight antibody can be constructed, and these genes can be introduced into a expression vector and then expressed in an appropriate host cell (for example, see Co et al., J. Immunol. (1994) 152: 2968). -76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63; Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).

The "scFv" is a single-chain polypeptide that binds two variable regions through a linker or the like as necessary. The two variable regions included in the scFv are usually 1 VH and 1 VL, but they can also be 2 VH or 2 VL. In general, scFv polypeptides include a linker between the VH and VL functional regions, thereby forming a paired portion of VH and VL necessary for antigen binding. Generally, in order to form a pairing moiety between VH and VL in the same molecule, in general, the linker linking VH and VL is set to a peptide linker of 10 or more amino acids in length. However, the linker of scFv in the present invention is not limited to the peptide linker as long as it does not hinder the formation of scFv. For the generalization of scFv, please refer to Pluckthun "The Pharmacology of Monoclonal Antibody" Vol.113 (Rosenburg and Moore ed., Springer Verlag, NY, pp. 269-315 (1994)).

In addition, "diabody (Db)" refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 ( 1993), EP404,097, WO93 / 11161, etc.). A bifunctional antibody is a dimer composed of two polypeptide chains, each of which has a short degree of inability to bind the light chain variable region (VL) and heavy chain variable region (VH) to each other in the same chain, such as 5 Linkers around each residue. Due to the short linker between VL and VH encoded on the same polypeptide chain, the single-chain V region fragment cannot be formed to form a dimer, so that the bifunctional antibody has two antigen-binding sites. At this time, if the combination of VL and VH and VLa-VHb and VLb-VHa of two different epitopes (a, b) is simultaneously expressed by a linker of about 5 residues, it will be double-specific Sexual Db is secreted. In this case, the two different epitopes may refer to an epitope at a different 2 position on the same antigen, or may refer to an epitope at a 2 position of the 2 different antigens, respectively.

Since a bifunctional antibody contains two molecules of scFv, it includes four variable regions. As a result, it has two antigen-binding sites. Unlike the case where scFv does not form a dimer, and for the purpose of forming a bifunctional antibody, usually the linker linking VH and VL in each scFv molecule is a peptide linker, which is determined to be 5 amine groups. Acid around. However, the peptide linker that forms the scFv of the bifunctional antibody is not limited to the linker as long as it does not hinder the performance of the scFv and does not prevent the formation of the bifunctional antibody.

Preferred polypeptides or heteromultimers for use in the method of the present invention include, for example, polypeptides or heteromultimers having a variable region of a heavy chain and a variable region of a light chain of an antibody. Polymer. Furthermore, a more preferred aspect of the present invention is an assembly control method, wherein the polypeptide or heteromultimer of the present invention includes two or more heavy chain variable regions and two or more light chain variable regions. The polypeptide or heteromultimer is preferably one capable of recognizing two or more epitopes, for example, having multiple specific antibodies.

In the present invention, multispecific antibodies are more preferred, such as bispecific antibodies.

That is, the preferred aspect of the present invention relates to a method for controlling assembly, which is controlled by, for example, two types of heavy chain variable regions (first heavy chain and second heavy chain) and two types of light chain variable regions (first Light chain and second light chain) assembly of bispecific antibodies.

If the “bispecific antibody” in a preferred aspect of the present invention is further detailed, the “first heavy chain” refers to the H chain forming one of the two H chains of the antibody, and the second H chain refers to the The first H chain is different from another H chain. That is, any one of the two H chains can be used as the first H chain and the other can be used as the second H chain. Similarly, the "first light chain" refers to the L chain that forms one of the two L chains of a bispecific antibody, and the second L chain refers to another L chain that is different from the first L chain. Any one of the L chains serves as the first L chain and the other serves as the second L chain. Generally, the first L chain and the first H chain are derived from the same antibody that recognizes an antigen (or epitope), and the second L chain and the second H chain are also derived from the same antibody that recognizes an antigen (or epitope). . Here, the L chain-H chain pair formed by the first H chain ‧ L chain is referred to as a first pair, and the L chain-H chain pair formed by the second H chain ‧ L chain is referred to as a second pair. It is preferred that the antigen (or epitope) used in the production of the second pair of antibodies is different from the user of the antibody in the first pair. That is, the first and second recognized antigens may be the same, However, it is preferred to recognize a different antigen (or epitope). In this case, it is preferable that the H and L chains of the first and second pairs have different amino acid sequences from each other. When the first pair and the second pair recognize different epitopes, the first pair and the second pair can recognize completely different antigens, and can also recognize different positions on the same antigen (different epitopes) ). One of them can recognize antigens such as proteins, peptides, genes, and sugars, while the other can recognize radioactive substances, chemotherapeutic agents, and cell-damaging substances such as cell-derived toxins. However, when considering the production of an antibody pair consisting of a specific H chain and L chain, the specific H chain and L chain can be arbitrarily determined as the first pair and the second pair.

The "bispecific antibody" is not necessarily limited to an antibody composed of two types of heavy chains and two types of light chains. For example, the two types of heavy chain variable regions and two types of light chain variable regions may be single-chain. Formally linked antibodies (eg sc (Fv) 2).

In the method of the present invention, a gene encoding an H chain or an L chain of an antibody before mutation introduction (in this specification, it may be separately described as "the antibody of the present invention") may use a known sequence or use the field Obtained by methods known to the public. For example, it can be obtained from an antibody library, or a gene encoding an antibody can be cloned from a fusion antibody producing a single antibody.

Regarding antibody libraries, there are already many well-known antibody libraries, and methods for making antibody libraries are also well known. Therefore, those skilled in the art can appropriately obtain antibody libraries. For example, for antibody phage libraries, refer to Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res .1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 3245-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14 and Japanese Special Table Hei 20 -504970 and other documents. In addition, a known method such as a method for preparing a eukaryotic cell bank (WO95 / 15393 pamphlet) or a liposome reminder method can be used. Moreover, techniques for obtaining human antibodies using panning are also known using human antibody libraries. For example, the variable region of a human antibody can be used as a single chain antibody (scFv), displayed on the surface of a phage by a phage display method, and a phage that binds to an antigen can be selected. If the selected phage is genetically analyzed, a DNA sequence encoding a variable region of a human antibody that binds to an antigen can be determined. If the DNA sequence of the scFv bound to the antigen is clear, an appropriate expression vector can be produced based on the sequence, and a human antibody can be obtained. These methods are well known and reference can be made to WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388.

A method for obtaining a gene encoded as an antibody from a fusion tumor can be obtained by basically using a known technique, using a desired antigen or a cell expressing the desired antigen as a sensory antigen, and immunizing these in accordance with a common immunological method. Immune cells are fused with well-known mother cells using a common cell fusion method, and single antibody-producing cells (fusion tumors) are screened using a usual screening method to obtain the mRNA of the fusion tumor using reverse transcriptase to synthesize the variable region of the antibody (V CDNA) obtained by ligating these to a DNA encoding a desired antibody constant region (C region).

More specifically, it is not particularly limited to those exemplified below, but is used to obtain a sensory antigen encoding the antibody genes encoding the H chain and the L chain, including a complete antigen with immunogenicity and a hapten including no immunogenicity. (hapten) both incomplete antigens. For example, a full-length protein or a partial peptide of a protein of interest can be used. In addition, substances composed of polysaccharides, nucleic acids, lipids, etc. are known to become Antigen, but the antigen of the antibody of the present invention is not particularly limited. The preparation of the antigen can be performed according to a method known to those skilled in the art, for example, a method using a baculovirus (for example, WO98 / 46777, etc.). The fusion tumor can be produced according to, for example, the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46) and the like. When the immunogenicity of the antigen is low, it can be bound to a large molecule with immunogenicity such as albumin and immunized. In addition, if necessary, the antigen can become a soluble antigen by binding to other molecules. When a membrane penetrating molecule such as a receptor is used as the antigen, the extracellular region portion of the receptor may be used as a fragment, or a cell expressing a membrane penetrating molecule on the cell surface may be used as an immunogen.

Antibody-producing cells can be obtained by immunizing an animal using the above-mentioned suitable sensory antigen. Alternatively, antibody-producing lymphocytes can be immunized in vitro to produce antibody-producing cells. Various mammals can be used for the immunized animal, and rodents, rabbits, and primates are generally used. For example, primates such as rodents such as mice, rats, hamsters, rabbits such as rabbits, macaca fascicularis, macaca mulatta, papio hamadryas, and chimpanzee animal. In addition, transgenic animals with a human antibody gene repertoire are also known, and human antibodies can also be obtained by using such animals (see WO96 / 34096; Mendez et al., Nat. Genet. 1997, 15 : 146-56). Instead of using the gene transgenic animal, for example, human lymphocytes can be sensed in vitro with a desired antigen or a cell expressing the desired antigen, and then the sensed lymphocytes can be used with human myeloma cells such as U266 fusion can also obtain the desired human antibody with binding activity to the antigen (see Japanese Patent Publication No. 1-59878). Also, you can A transgenic animal having repertoires possessing all human antibody genes is immunized with a desired antigen to obtain a desired human antibody (see WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, WO96 / 33735).

Immunization of animals can be performed, for example, by appropriately diluting and suspending the sensation antigen with Phosphate-buffered Saline (PBS) or physiological saline, and then mixing with an adjuvant and emulsifying as needed. This is done intraperitoneally or subcutaneously. Thereafter, it is preferable to administer the sensory antigen mixed with Freud's incomplete adjuvant several times at intervals of 4 to 21 days. Confirmation of antibody production can be performed by measuring the target antibody titer in animal serum using conventional methods.

Fusion tumors can be produced by fusing antibody-producing cells from animals or lymphocytes that have been immunized with the desired antigen, using conventional fusion agents (such as polyethylene glycol) and myeloma cells (Goding, Monoclonal Antibodies: Principles and Practice Academic Press, 1986, 59-103). If necessary, the fused tumor cells are cultured and proliferated, and the specificity of the antibodies produced by the fused tumor is determined using known methods such as immunoprecipitation, radioimmunoassay (RIA), and enzyme-linked immunosorbent analysis (ELISA). . Thereafter, if necessary, fusion tumors that produce antibodies with specificity, affinity, or activity for the purpose of the measurement may be sub-cloned by methods such as limiting dilution.

Next, a gene encoding the selected antibody is used to probe the antibody specifically (e.g., an oligonucleotide complementary to the sequence encoding the constant region of the antibody) from the fusion tumor or antibody-producing cell (sense Lymphocytes, etc.). In addition, it is also possible to perform colonization from mRNA by RT-PCR. Immunoglobulins are classified into 5 different classes of IgA, IgD, IgE, IgG, and IgM. Furthermore, these categories are divided into several subtypes (isotypes) (e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2, etc.). In the present invention, the H chain and L chain used for antibody production may be derived from antibodies belonging to any of these types and subtypes. Although not particularly limited, IgG is particularly preferred.

Here, the genes encoding the H chain and the L chain can also be changed using genetic engineering methods. For example, for antibodies such as mouse antibodies, rat antibodies, rabbit antibodies, hamster antibodies, sheep antibodies, and camel antibodies, genetically modified antibodies, such as chimeric antibodies, that have been artificially altered to reduce heterogeneous antigenicity to humans can be appropriately prepared. , Humanized antibodies, etc. Chimeric antibodies are antibodies composed of mammals other than humans, such as the variable regions of the H and L chains of mouse antibodies, and the constant regions of the H and L chains of human antibodies. The DNA of the variable region of the antibody is linked to the DNA encoding the invariant region of the human antibody, and these are inserted into the expression vector and introduced into the host for generation. Humanized antibodies, also known as reshaped human antibodies, can be designed using DNA sequences designed to link complementary complementarity determining regions (CDRs) of mammals other than humans, such as mouse antibodies. Several oligonucleotides prepared with overlapping portions at the ends were synthesized by the PCR method. It can be obtained by linking the obtained DNA with a DNA encoding an invariant region of a human antibody, and then embedding it into a expression vector, introducing them into a host and producing them (see EP239400; WO96 / 02576). Among the FRs of human antibodies linked via CDRs, those in which the complementarity determining region forms a good antigen-binding site are selected. If necessary, the amino acid of the framework region of the antibody variable region among the complementarity determining regions of the reconstituted human antibody may also be substituted to form an appropriate antigen binding site (K. Sato et al., Cancer Res. 1993 53: 851-856).

In addition to the above-mentioned humanization, for example, The biological properties of antibodies such as binding are changed. Such changes can be performed using site-specific mutations (for example, refer to Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488), PCR mutation, cassette mutation and other methods. Generally speaking, compared with the amino acid sequence of the variable region of the original antibody, the antibody variant with improved biological characteristics has more than 70%, preferably 80% or more, and more preferably 90% or more (for example, 95% or more, 97%, 98%, 99%, etc.) amino acid sequence identity and / or similarity. In this specification, sequence identity and / or similarity is defined as: the sequence of the sequence is maximized to obtain the maximum sequence identity and the gap is introduced into the gap, and the residue is the same as the original antibody residue ( Identical residues) or similar (generally, amino acid residues classified into groups based on the characteristics of the amino acid side chains) of the amino acid residues. Generally, natural amino acid residues are classified into the following groups based on the nature of their side chains: (1) Hydrophobicity: alanine, isoleucine, n-leucine, valine, methionine, and white Glycine; (2) neutral hydrophilicity: asparagine, glutamine, cysteine, threonine, and serine; (3) acidity: aspartic acid and glutamic acid; (4) ) Basic: arginine, histidine, and lysine; (5) residues affecting chain alignment: glycine and proline; and (6) aromaticity: tyrosine, tryptophan, and Phenylalanine.

In general, a total of six complementarity determining regions (hypervariables; CDRs) present in the variable regions of the H and L chains interact and form the antigen-binding site of the antibody. Among them, it is known that although one variable region has a lower affinity than a person including a complete binding site, it recognizes an antigen and has a binding ability. Therefore, the antibody genes encoded by the H chain and L chain in the present invention need only maintain the binding of the polypeptide encoded by the gene to the desired antigen, and can encode the fragments including the antigen binding sites of the H chain and the L chain. Part of it.

According to the assembly control method of the present invention, as described above, for example, the desired bispecific antibody can be obtained preferentially (efficiently). That is, bispecific antibodies can be efficiently formed from the monomer mixture into the desired heteromultimer.

Hereinafter, the case of an IgG type bispecific antibody having two types of heavy chain variable regions (VH1 and VH2) and two types of light chain variable regions (VL1 and VL2) will be described in more detail, but other heteromultimers are also described. The method of the present invention is equally applicable.

To obtain an epitope recognizable from one of the first heavy chain variable region (VH1) and the first light chain variable region (VL1), and the second heavy chain variable region (VH2) and the second light chain The chain variable region (VL2) recognizes the situation of a bispecific antibody with another epitope. If the production of the antibody is performed with 4 different chains, it is theoretically possible to produce 10 antibody molecules.

In this case, if controlled in a manner that inhibits, for example, the assembly between VH1 and VL2, and / or VH2 and VL1 polypeptides, the desired antibody molecule can be preferentially obtained.

For example, by changing the amino acid residues forming the interface between the VH1 polypeptide and the VL2 polypeptide and / or the VH2 polypeptide and the VL1 polypeptide as described above, the assembly between the polypeptides can be inhibited.

In addition, by using the assembly control method of the present invention, the assembly of heavy chains (VH1 and VH2) or light chains (VL1 and VL2) can be suppressed.

The heavy chain variable region is generally composed of three CDR regions and FR regions as described above. In the preferred aspect of the present invention, the amino acid residue for "change" can be appropriately selected from, for example, amino acid residues located in the CDR region or the FR region. In general, changes in amino acid residues in the CDR regions may Reduced binding capacity. Therefore, although the amino acid residue to be "modified" in the present invention is not particularly limited, it is preferably selected appropriately from the amino acid residues located in the FR region.

If it is a person skilled in the art, regarding the desired polypeptide to be controlled for assembly by the method of the present invention, it can be appropriately understood that it is the kind of amino acid residue in the FR interface which is close to the assembly.

Furthermore, among humans, mice, and other organisms, sequences that are FRs of the variable regions of antibodies can be used, and those skilled in the art can appropriately obtain them using public databases and the like. More specifically, the amino acid sequence information of the FR region can be obtained by using a tool of the embodiment described later.

For example, regarding the bispecific antibodies shown in the examples described below, specific examples of amino acid residues near the FR interface during assembly include, for example, position 39 (FR2 region) on the variable region of the heavy chain (for example, Sequence number: 39 in amino acid sequence of 6) glutamine (Q), and position 38 (FR2 region) on the variable region of the light chain (contact) (e.g., amine of sequence number: 8) Glutamine (Q) at position 44 in the amino acid sequence). In addition, for example, it is preferably leucine (L) at position 45 (FR2) on the variable region of the heavy chain (e.g., position 45 of the amino acid sequence of sequence number 6), and relatively light chain variable Proline (P) at position 44 (FR2) on the region (for example, position 50 of the amino acid sequence of 8). For the numbering of these parts, refer to the literature by Kabat et al. (Kabat EA et al. 1991. Sequence of Proteins of Immunological Interest. NIH).

As shown in the examples described later, by changing the amino acid residues and carrying out the method of the present invention, the desired antibody can be preferentially obtained.

Since these amino acid residues are known to be high in humans and mice Conservative (J.Mol.Recognit. 2003; 16: 113-120). Therefore, the assembly of VH and VL other than the antibodies shown in the examples can also be changed by corresponding to the amino acid residues. Amino acid residues, and control the assembly of variable regions of the antibody.

That is, a preferred aspect of the present invention is to provide an antibody that is an antibody (polypeptide (e.g., sc (Fv) 2)) or heteromultimer (e.g., IgG-type antibody) comprising a heavy chain variable region and a light chain variable region. Etc.), the following amino acid residues (1) and (2) or (3) and (4) are amino acid residues with the same charge:

(1) An amino acid residue, contained in the variable region of the heavy chain, corresponding to position 39 in the amino acid sequence of SEQ ID NO: 6.

(2) An amino acid residue, which is contained in the variable region of the light chain and corresponds to position 44 in the amino acid sequence of SEQ ID NO: 8.

(3) An amino acid residue included in the variable region of the heavy chain, corresponding to position 45 in the amino acid sequence of SEQ ID NO: 6.

(4) An amino acid residue, which is contained in the variable region of the light chain and corresponds to the 50th position in the amino acid sequence of SEQ ID NO: 8.

The amino acid sequence of the above sequence number: 6 or 8 is used to more specifically exemplify the position of the amino acid residue to be changed in the present invention, and the heavy chain variable region or light chain variable region is not limited to this. In the case of amino acid sequences.

Each of the above amino acid residues (1) and (2), (3), and (4), as shown in the examples described below and FIG. 1, are close to each other after assembly. As long as it is a person in the technical field, the same degree simulator using commercially available software can be used for the desired heavy chain variable region or light chain variable region to find the amine group similar to (1) to (4) above. The position corresponding to the acid residue, and the amino acid residue at the position is appropriately changed.

Among the above antibodies, the "amino acid residue having a charge" is preferably selected from, for example, amino acid residues contained in any of the following groups (a) or (b).

(a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), and histidine (H).

Furthermore, the present invention provides an antibody which is an antibody (polypeptide or heteromultimer, etc.) comprising a variable region of a heavy chain and a variable region of a light chain, and any one of the following (3) and (4) is a charged amine Acid residues. The side chains of the amino acid residues represented by (3) and (4) below can approach and form a hydrophobic core.

(3) An amino acid residue included in the variable region of the heavy chain, corresponding to position 45 in the amino acid sequence of SEQ ID NO: 6.

(4) An amino acid residue, which is contained in the variable region of the light chain and corresponds to the 50th position in the amino acid sequence of SEQ ID NO: 8.

Among the above antibodies, the "charged amino acid residue" is preferably, for example, glutamic acid (E), aspartic acid (D), lysine (K), arginine (R) or a group Amino acid (H).

The amino acid residues of (1) to (4) above are usually (1) glutamine (Q), (2) glutamine (Q), and (3) leucine in humans and mice, respectively. (L), (4) Proline (P). Therefore, in a preferred aspect of the present invention, the amino acid residues are provided for alteration (for example, substituted with a charged amino acid). The types of the amino acid residues (1) to (4) are not necessarily limited to the amino acid residues described above, and may be other amino acids equivalent to the amino acid residues. For example, the amino acid corresponding to position 44 in the amino acid sequence of the sequence number: 8 on the variable region of the light chain, in the case of humans, may be, for example, histidine (H). Those skilled in the art can refer to known literatures (for example, J. Mol. Recognit. 2003; 16: 113-120), and can assume the post number: 8 The desired position is known, and the type of amino acid residue corresponding to the position is known, and the amino acid residue is appropriately changed (for example, substituted with a charged amino acid).

In addition, the method for producing the above-mentioned antibody and the assembly control method of the present invention in which the above-mentioned (1) to (4) amino acid residues are changed to specific features are also preferred aspects of the present invention.

In another aspect of the present invention, for example, there is a method of introducing a charge repulsive force at the interface of a heavy or light chain constant region and suppressing the assembly of heavy chains with each other or between the heavy and light chains. Amino acid residues contacted at the interface of the heavy chain invariant region, for example, relative to the 377 position (356 position) and 470 position (439 position), 378 position (357 position), and 393 position (370 position) in the CH3 region , 427 (399) and 440 (409) areas. The amino acid residue in contact with the interface between the heavy chain invariant region and the light chain invariant region includes, for example, a region opposite to the 221 position (213 position) of the CH1 region and the 123 position of the CL region. As for the numbering of the invariant regions of the antibody, reference is made to Kabat et al. (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH). The EU numbering of the invariant regions of the heavy chain is shown in parentheses.

As shown in the examples described later, by changing these amino acid residues and implementing the method of the present invention, the assembly of the heavy chain of the antibody can be controlled, and the desired antibody can be preferentially obtained.

That is, a preferred aspect of the present invention provides an antibody comprising two or more heavy chain CH3 region antibodies and an Fc region binding protein (eg, an IgG antibody, minibody (Alt M et al. FEBS Letters 1999; 454: 90- 94), immunoadhesin (non-patent document 2), etc.), among the CH3 region of the first heavy chain, selected from the group consisting of amino acid residues shown in (1) to (3) below Group to 3 Histidine residues have the same charge.

(1) An amino acid residue, which is contained in the CH3 region of the heavy chain, and is numbered at positions 356 and 439 in the EU.

(2) An amino acid residue, which is contained in the CH3 region of the heavy chain, and is numbered 357 and 370 by the EU number.

(3) An amino acid residue, which is contained in the CH3 region of the heavy chain, and is numbered 399 and 409 at the EU number.

Furthermore, in a preferred aspect of the present invention, there is provided an antibody selected from the amino acid residues shown in the above (1) to (3) among the second heavy chain CH3 regions that are different from the first heavy chain CH3 regions. A group of amino acid residues in the group of groups corresponding to the group of amino acid residues shown in the above (1) to (3) with the same charge in the first heavy chain CH3 region The three amino acid residues have the opposite charge in the corresponding amino acid residue in the CH3 region of the first heavy chain.

Each of the above amino acid residues (1) to (3) is as shown in the example described later and FIG. 27, and approaches each other after assembly. For those in the technical field, for the desired heavy chain CH3 region or heavy chain constant region, by using the same degree simulator of commercially available software, the amino acid residues corresponding to the above (1) to (3) can be found And the amino acid residue of the site is appropriately changed.

Among the above antibodies, the "charged amino acid residue" is preferably, for example, selected from amino acid residues contained in any of the following groups (a) or (b).

(a) glutamic acid (E), aspartic acid (D); (b) lysine (K), arginine (R), and histidine (H).

In the above antibodies, "having the same kind of charge" means, for example, that any one of two or more amino acid residues has an amine contained in any one of the groups (a) or (b) above. Acid residues. "Reversely charged" means, for example, a case where at least one amino acid residue among two or more amino acid residues has the amino acid residue contained in any one of the groups (a) or (b) above, The remaining amino acid residues have amino acid residues contained in different groups.

In a preferred aspect, among the above antibodies, the first heavy chain CH3 region and the second heavy chain CH3 region can be crosslinked by a disulfide bond.

In the present invention, the amino acid residue for "changing" is not limited to the amino acid residue in the variable region or the constant region of the antibody. As long as it is a person skilled in the art, for polypeptide variants or heteromultimers, by using the same degree simulator of commercially available software, etc., the amino acid residues forming the interface can be found, and the amino acid at the site can be found. Residues are provided for change to control assembly.

The method of the present invention can be implemented in combination with known techniques, but it is not necessary. For example, in order to promote the assembly of VH1 and VL1, and / or VH2 and VL2, in addition to the "change" of the present invention, the amino acid side chain existing in one of the variable regions of the H chain is replaced with a larger one Side chain (knob; protrusion), and the amino acid side chain existing in the relatively variable region of another H chain is replaced with a smaller side chain (hole; gap), and the protrusion can be arranged in the gap to promote The assembly of VH1 and VL1 and / or VH2 and VL2 can further inhibit the assembly between VH1 and VL2, and / or VH2 and VL1 polypeptides.

The assembly control method of the present invention can be appropriately implemented when the desired sc (Fv) 2 is obtained preferentially (efficiently). In the following, the case of sc (Fv) 2 having two types of heavy chain variable regions (H1 and H2) and two types of light chain variable regions (L1 and L2) will be described in more detail.

In general, the sc (Fv) 2 line divides two heavy chain variable regions (VH1 A single-chain polypeptide that binds to VH2) and two light chain variable regions (VL1 and VL2) with linkers. That is, sc (Fv) 2 is a low-molecular-weight antibody that binds four antibody variable regions with a linker or the like to form a single chain. Generally, sc (Fv) 2 is a single-chain antibody that combines four variable regions, such as two light chain variable regions and two heavy chain variable regions, with a linker or the like (Hudson et al, J Immunol. Methods 1999; 231: 177-189).

Those skilled in the art can make sc (Fv) 2 by a known method, for example, they can be produced by linking scFv with a linker. scFv contains VH and VL of the antibody. These regions exist in a single polypeptide chain (for a general discussion of scFv, refer to Pluckthun "The Pharmacology of Monoclonal Antibodies" Vol. 113 (Rosenburg and Moore ed (Springer Verlag, New York) pp. 269-315, 1994)).

In addition, the antibody is preferably such that two VH and two VL are based on the N-terminal side of the single-chain polypeptide, and are sequentially connected by VH, VL, VH, and VL ([VH] linker [VL] linker [VH] Child [VL]).

The order of the two VHs and the two VLs is not particularly limited to the above configuration, and they can be arranged in any order. For example, it may be configured as follows.

[VL] linker [VH] linker [VH] linker [VL]

[VH] linker [VL] linker [VL] linker [VH]

[VH] linker [VH] linker [VL] linker [VL]

[VL] linker [VL] linker [VH] linker [VH]

[VL] linker [VH] linker [VL] linker [VH]

sc (Fv) 2 may include an amino acid sequence other than the variable region of the antibody and the linker.

The variable region of the antibody may be the entire length of the variable region, as long as it can maintain the binding activity to the antigen, or it may be a partial sequence of the variable region. The amino acid sequence in the variable region may be substituted, deleted, added, inserted, or the like. For example, in order to reduce antigenicity, chimerization or humanization may be performed.

As the linker that binds to the variable region of the antibody, any peptide linker or synthetic compound linker introduced by genetic engineering (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used. However, a peptide linker is preferred in the present invention. The length of the linker is not particularly limited, and may be appropriately selected by those skilled in the technical field according to the purpose, but the preferred length is 12 amino acids or more (although the upper limit is not particularly limited, usually 30 amino acids or less, preferably 20) Less than amino acids), particularly preferably 15 amino acids. In the case where sc (Fv) 2 contains three peptide linkers, all peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.

For example, in the case of a peptide linker, for example: Ser

Gly‧Ser

Gly‧Gly‧Ser

Ser‧Gly‧Gly

Gly‧Gly‧Gly‧Ser

Ser‧Gly‧Gly‧Gly

Gly‧Gly‧Gly‧Gly‧Ser

Ser‧Gly‧Gly‧Gly‧Gly

Gly‧Gly‧Gly‧Gly‧Gly‧Ser

Ser‧Gly‧Gly‧Gly‧Gly‧Gly

Gly‧Gly‧Gly‧Gly‧Gly‧Gly‧Ser

Ser‧Gly‧Gly‧Gly‧Gly‧Gly‧Gly

(Gly‧Gly‧Gly‧Gly‧Ser) n

(Ser‧Gly‧Gly‧Gly‧Gly) n

[N is an integer of 1 or more] and the like. However, those skilled in the art can appropriately select the peptide linker length or sequence depending on the purpose.

A preferred aspect of sc (Fv) 2 is, for example, the following sc (Fv) 2.

[VH] linker (15 amino acids) [VL] linker (15 amino acids) [VH] linker (15 amino acids) [VL]

Synthetic chemical linkers (chemical cross-linking agents) are commonly used cross-linking agents in the cross-linking of peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), Di (thiosuccinimide) suberate (BS ³ ), dithiobis (succinimide propionate) (DSP), dithiobis (thiosuccinimide propionate) ( DTSSP), ethylene glycol bis (succinimide iminosuccinate) (EGS), ethylene glycol bis (succinimide iminosuccinate) (thio-EGS), disuccinimide tartarate (DST), dithiosuccinimide imino tartrate (sulfur-DST)), bis [2- (succinimide oxycarbonyloxy) ethyl] fluorene (BSOCOES)), bis [2- (thiosuccinic acid) Imineoxycarbonyloxy) ethyl] fluorene (sulfur-BSOCOES), etc., such crosslinking agents are commercially available.

When four variable regions of an antibody are bound, three linkers are usually required. The same linker can be used for all or different linkers can be used.

Among the sc (Fv) 2, structural isomers include, for example, single-chain bifunctional antibody (diabody) type and bivalent scFv type.

sc (Fv) 2 uses [variable region 1] (linker 1) [variable region 2] (connected Adaptor 2) [variable region 3] (linker 3) [variable region 4] is arranged in the order of bivalent scFv in the present invention: it has variable region 1 and variable region 2 Sc (Fv) 2 of the structure in which the variable region 3 and the variable region 4 are assembled. In the present invention, a single-chain bifunctional antibody (diabody) refers to sc (Fv) 2 having a structure in which variable region 1 and variable region 4 are assembled, and variable region 2 and variable region 3 are assembled.

The single-chain bifunctional antibody (diabody) has, for example, sc (Fv) 2 having a structure on the right in FIG. 12 (b), and the bivalent scFv type has, for example, sc (Fv) 2 having a structure on the left in FIG. 12 (b).

Whether sc (Fv) 2 is a single-chain bifunctional antibody (diabody) type or a bivalent scFv type can be analyzed by, for example, a protease-limited decomposition method. For example, the following method can be used for analysis.

The subunit of sc (Fv) 2 was subtilisin A, a subtilisin protease that can be partially and limitedly decomposed to perform the limited decomposition of sc (Fv) 2.

In the case of a single-chain bifunctional antibody (diabody) type, due to the interaction between VH and VL, among the three linkers of sc (Fv) 2, the molecular weight is observed even if any of the linkers is cut. No change.

On the other hand, in the case of the bivalent scFv type, if the central linker is cut off, a molecular species with a half molecular weight will be generated.

Therefore, by analyzing the reaction product, a bivalent scFv type can be distinguished from a single-chain bifunctional antibody type.

The reaction product can be analyzed by, for example, gel filtration chromatography analysis. Alternatively, depending on the area of the peak, chromatography can be used to determine the bivalence included in sc (Fv) 2. The ratio of (bivalent) sc (Fv) 2 structure to single-chain bifunctional antibody (diabody) structure was quantitatively evaluated.

When the assembly control method of the present invention is to obtain the desired sc (Fv) 2 preferentially, that is, one of a bifunctional antibody (diabody) type and a bivalent scFv type, it is more preferable. Best available.

More specifically, when sc (Fv) 2 has a structure of VH1- (linker) -VL1- (linker) -VH2- (linker) -VL2, using the assembly control method of the present invention, it is preferred to obtain In the case of bivalent scFv-type sc (Fv) 2, for example, it is only necessary to suppress the assembly of VH1 and VL2, and / or VH2 and VL1. (For example, a mutation is introduced at the interface between VH1 and VL2, so that the amino acid residue becomes the same kind of charge.)

When sc (Fv) 2 of a single-chain bifunctional antibody (diabody) type is preferentially obtained, it is only necessary to inhibit the assembly of, for example, VH1 and VL1, and / or VH2 and VL2. (For example, a mutation is introduced at the interface between VH1 and VL1, so that the amino acid residue becomes the same kind of charge.)

When the sc (Fv) 2 is a monospecific antibody, the present invention can be implemented in the same manner.

Moreover, in addition to these techniques, the functional regions of VH and VL can also be cross-linked with disulfide bonds (Clin Cancer Res. 1996 Feb; 2 (2): 245-52).

By using the assembly control method of the present invention, for example, an active antibody or polypeptide can be efficiently produced. Examples of the activity include binding activity, neutralizing activity, cytotoxic activity, synergistic activity, antagonistic activity, and enzyme activity. Synergistic activity refers to the activity that induces changes in certain physiological activities by binding antibodies to antigens such as receptors and carrying out signal transmission in cells. Physiological activity For example: proliferation activity, survival activity, differentiation activity, replication activity, membrane transport activity, binding activity, proteolytic activity, phosphorylation / dephosphorylation activity, redox activity, transfer activity, nucleic acid decomposition activity, dehydration activity, cell death Inducing activity, apotosis inducing activity, and the like are not limited thereto.

In addition, by the method of the present invention, antibodies or polypeptides that recognize a desired antigen or bind to a desired receptor can be efficiently produced.

The antigen is not particularly limited, and may be any antigen. Examples of the antigen include, for example, a receptor or a fragment thereof, a cancer antigen, an MHC antigen, a differentiation antigen, and the like, and are not particularly limited to these.

Examples of the receptor include receptors belonging to the following receptor families: hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase Receptor family, TNF receptor family, G protein conjugate receptor family, GPI anchor receptor family, tyrosine phosphatase receptor family, adhesion factor family, hormone receptor family Wait. Receptors belonging to these receptor families and most of their literature exist, for example: Cooke BA., King RJB., Van der Molen HJ.ed. New Comprehesive Biochemistry Vol. 18B "Hormones and their Actions Part II" pp .1-46 (1988) Elsevier Science Publishers BV., New York, USA, Patthy L. (1990) Cell, 61: 13-14., Ullrich A., et al. (1990) Cell, 61: 203-212 ., Massagul J. (1992) Cell, 69: 1067-1070., Miyajima A., et al. (1992) Annu. Rev. Immunol., 10: 295-331., Taga T. and Kishimoto T. (1992 ) FASEB J., 7: 3387-3396., Fantl WI., Et al. (1993) Annu. Rev. Biochem., 62: 453-481., Smith CA., et al. (1994) Cell, 76: 959-962., Flower DR. (1999) Biochim. Biophys. Acta, 1422: 207-234., Review by Miyasaka Masaaki, Cell Engineering Special Booklet Series "Adhesion Factor Handbook" (1994) (Shurunsha, Tokyo, Japan) For specific receptors belonging to the aforementioned receptor family, for example: human or mouse erythropoietin (EPO) receptor, human or mouse colony stimulating factor (G-CSF) receptor, human or Mouse thrombopoietin (TPO) receptor, human or mouse insulin receptor, human or mouse Flt-3 ligand receptor, human or mouse platelet derived proliferation factor (PDGF) receptor, human or mouse interference (IFN) -α, β receptor, human or mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) -10 receptor , Human or mouse insulin-like proliferation factor (IGF) -I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse hair body neurotrophic factor (CNTF) receptor, etc. (hEPOR: Simon , S. et al. (1990) Blood 76, 31-35 .; mEPOR: D'Andrea, AD. Et al. (1989) Cell 57,277-285 .; hG-CSFR: Fukunaga, R. et al. (1990 ) Proc.Natl.Acad.Sci.USA.87,8702-8706 .; mG-CSFR: Fukuunaga, R. et al. (1990) Cell 61,341-350 .; hTPOR: Vigon, I.et al. (1992) 89,5640-5644 .; mTPOR: Skoda, RC.Et al. (1993) 12, 2645-2653 .; hInsR: Ullr ich, A. et al. (1985) Nature 313, 756-761 .; hFlt-3: Small, D. et al. (1994) Proc. Natl. Acad. Sci. USA. 91, 459-463 .; hPDGFR: Gronwald, RGK Et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 3435-3439 .; hIFNα / βR: Uze, G. et al. (1990) Cell 60, 225-234. And Novik, D.et al (1994) Cell 77, 391-400.).

Cancer antigens are antigens that appear with the deterioration of cells, and they also become tumor-specific antigens. Also, when a cell is cancerous, it is on the cell surface or on a protein molecule The abnormal sugar chains that appear also become cancer antigens, especially called cancer sugar chain antigens. Examples of cancer antigens include CA19-9, CA15-3, and Serail SSEA-1 (SLX).

MHC antigens are roughly divided into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, -H, and MHC class II antigens. Contains HLA-DR, -DQ, -DP.

Differentiation antigens include CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD23, CD25, CD28, CD29 , CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55 , CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD71, CD73, CD95, CD102, CD106, CD122, CD126, CDw130 and so on.

The present invention also provides a polypeptide variant or heteromultimer whose assembly is controlled by the method of the present invention. That is, the present invention relates to a polypeptide or heteromultimer obtained by the assembly control method of the present invention.

A preferred aspect of the present invention is to provide a polypeptide variant in which the amino acid residues forming the interface in the polypeptide are changed to inhibit assembly in the original polypeptide.

Furthermore, in another aspect of the present invention, a heteromultimer is provided. The amino acid residues forming the interface between the polypeptides are changed to inhibit the assembly between the original polypeptides.

In the present invention, "the original polypeptide" refers to the use of the method of the present invention. A polypeptide that changes its former state in a controlled manner.

An example of the above-mentioned polypeptide variants of the present invention, for example, the original polypeptide can form two kinds of structural and structural isomers. In addition, as an example of the aforementioned heteromultimer, for example, the original polypeptide can form a multimer of two or more kinds of multimers.

In addition, a polypeptide variant or heteromultimer whose assembly is controlled by using the assembly control method of the present invention is also included in the present invention. That is, among the preferred aspects of the above assembly control method, the controlled assembly of the polypeptide or heteromultimer is also one of the preferred aspects of the present invention.

In addition, the present invention provides a method for manufacturing a polypeptide or a plurality of multimers whose assembly is controlled.

In a preferred aspect of the manufacturing method of the present invention, a method for manufacturing a polypeptide variant is provided, which is a method for manufacturing a polypeptide having a variation in amino acid residues forming an interface in the polypeptide in a controlled manner of polypeptide assembly, including the following steps: ( a) altering a nucleic acid encoding an amino acid residue forming an interface within a polypeptide from a pro-nucleic acid, thereby inhibiting internal assembly of the polypeptide, and (b) culturing the host cell to express the nucleic acid, and (c) from the host cell culture The polypeptide is recovered.

Furthermore, in another aspect of the manufacturing method of the present invention, a method for manufacturing a heteromultimer is provided, in which the amino acid residues forming the interface between the polypeptides are mutated in a manner that controls the assembly of the heteromultimers. A method for producing a polymer includes the following steps: (a) changing a nucleic acid encoding an amino acid residue forming an interface between polypeptides from an original nucleic acid, thereby suppressing the assembly between the polypeptides, and (b) cultivating a host cell for expression The nucleic acid, and (c) the heteromultimer is recovered from the host cell culture.

Furthermore, using the assembly control method of the present invention, The method of suppressing the method to change the nucleic acid encoding the amino acid residue forming the interface between (in) the polypeptide from the original nucleic acid is also one of the preferred aspects of the above-mentioned manufacturing method of the present invention.

"Altering a nucleic acid" in the above method of the present invention refers to altering a nucleic acid in a manner corresponding to an amino acid residue introduced corresponding to the "change" in the present invention. More specifically, it refers to a nucleic acid that encodes an amino acid residue that was originally (before alteration) changed to a nucleic acid that encodes an amino acid residue introduced by alteration. Generally, it means that the original nucleic acid is subjected to genetic manipulation or mutation treatment such as insertion, deletion or substitution of at least one base, so that it becomes a codon encoding an intended amino acid residue. That is, a codon encoded as an ortho amino acid residue is replaced with a codon encoded as an amino acid residue introduced as a result of the change. If the nucleic acid is changed, a person skilled in the art can use a known technique such as a site-specific mutation induction method, a PCR mutation introduction method, or the like to appropriately implement it.

In the present invention, the nucleic acid is usually carried (inserted) on an appropriate vector and introduced into a host cell. This vector is not particularly limited as long as it can stably retain the inserted nucleic acid. For example, if the host uses coliform, the selection vector is preferably a pBluescript vector (manufactured by Stratagene), and various commercially available vectors can be used. In the case where a carrier is used for producing the polypeptide of the present invention, it is particularly useful to express the carrier. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in a test tube, coliform, cultured cell, or organism. For example, if it is expressed in a test tube, a pBEST vector (promega) is preferred. A pET vector (manufactured by Invitrogen) is preferred, a pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred in cultured cells, and a pME18S vector (Mol Cell Biol. 8: 466-472 ( 1988)) and so on. The invention The DNA insertion vector can be carried out by a conventional method, for example, using a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

The host cell is not particularly limited, and various host cells can be used depending on the purpose. Cells used to express peptides, such as bacterial cells (eg, streptococcus, staphylococcus, coliform, streptomyces, subtilis), fungal cells (eg, yeast, pinworm), insect cells (eg, Drosophila S2, Twill Spodoptera SF9), animal cells (for example: CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes Melanoma cells) and plant cells. For introduction of a vector into a host cell, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), liposome ( Lipofectamine) method (manufactured by GIBCO-BRL), microinjection method and other known methods.

In order for the polypeptide expressed in the host cell to be secreted into the inner cavity of the microsomal body, the peripheral cavity of the cell or the extracellular environment, an appropriate secretion signal may be embedded in the polypeptide of interest. These signals can be endogenous or heterologous to the polypeptide of interest.

In the above manufacturing method, the polypeptide is recovered when the polypeptide of the present invention is secreted into the culture medium. When the polypeptide of the present invention is produced in a cell, the cell is lysed before the polypeptide is recovered.

When the polypeptide of the present invention is recovered and purified from a recombinant cell culture, it can be used with ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography. , Well-known methods of hydroxyapatite chromatography and lectin chromatography.

The present invention relates to a composition (medicine) comprising the polypeptide variant of the present invention or the heteromultimer of the present invention and a pharmaceutically acceptable carrier.

In the present invention, a medicinal composition refers to an agent that is generally used for the treatment or prevention or inspection and diagnosis of a disease.

Those skilled in the art can prepare the pharmaceutical composition of the present invention by a known method. For example, it can be used parenterally in the form of a sterile solution or suspension of water or other pharmaceutically acceptable liquid for injection. For example, with pharmacologically acceptable carriers or media, specifically, sterilized water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, carriers, preservatives Agents, binding agents, etc. are appropriately combined and formulated in the unit dosage form required by generally accepted pharmaceutical habits. Among these preparations, the amount of the active ingredient is set to an appropriate capacity to obtain the indicated range.

Sterile compositions for injection can be formulated using carriers such as distilled water for injection, in accordance with usual formulation practices.

Aqueous solutions for injection, for example: physiological saline, glucose or isotonic solutions containing other auxiliary drugs (such as D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate dissolution aids such as alcohols (such as ethanol), polyhydric alcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80 (TM), HCO-50, etc.) may be used in combination.

For the oily liquid, for example, sesame oil, soybean oil, and a dissolution aid such as benzyl benzoate and / or benzyl alcohol can be used in combination. In addition, buffering agents (e.g., phosphate buffer and sodium acetate buffer) and analgesics (e.g., procaine hydrochloride) may also be added. (procain)), stabilizers (eg, benzyl alcohol and phenol), antioxidants. The prepared injection solution is usually filled in a suitable injection solution bottle.

The pharmaceutical composition of the present invention is preferably administered orally. For example, the composition can be made into an injection form, a nasal administration form, a pulmonary administration form, or a transdermal administration form. For example, intravenous, intramuscular, intraperitoneal, and subcutaneous injections can be used for systemic or local administration.

The administration method can be appropriately selected according to the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be set, for example, in a range of 0.0001 mg to 1000 mg per 1 kg of body weight. Or, for example, a dose of 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The amount and method of administration vary depending on the patient's weight, age, symptoms, etc., as long as those in the technical field can consider these conditions and set the appropriate amount and method of administration.

If necessary, the polypeptide or heteromultimer of the present invention may be formulated by combining it with other pharmaceutical ingredients.

The present invention also provides a nucleic acid encoding a polypeptide variant of the present invention or a heteromultimer of the present invention. Furthermore, a vector carrying the nucleic acid is also included in the present invention.

Furthermore, the present invention provides a host cell having the above nucleic acid. The host cell is not particularly limited, and examples include coliform bacteria and various animal cells. The host cell can be used as a production line for producing or expressing the antibody or polypeptide of the present invention, for example. Production lines for peptide manufacturing include in vitro and in vivo production lines. The in vitro production system includes, for example, a production system using eukaryotic cells and a production system using prokaryotic cells.

Eukaryotic cells that can be used as host cells, such as animal cells, plant cells, and fungal cells. Animal cells, mammalian cells such as CHO (J.Exp.Med. (1995) 108: 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, etc .; amphiphilic cells such as Xenopus laevis eggs Mother cells (Valle et al., Nature (1981) 291: 338-340); and insect cells such as Sf9, Sf21, Tn5. For the performance of the antibody of the present invention, CHO-DG44, CHO-DX11B, COS7 cells, and BHK cells are preferably used. Among animal cells, CHO cells are particularly preferred for a large number of expressions. The introduction of a vector into a host cell can be performed by, for example, a calcium phosphate method, a DEAE glucan method, a method using a cationic liposome DOTAP (manufactured by Boehringer Mannheim), an electroporation method, and a lipofection method. Method.

As a plant cell, for example, a cell derived from Nicotiana tabacum is known as a protein production system, and the cell can be produced by using a healing tissue culture method to produce the antibody of the present invention. For fungal cells, yeasts are used, for example: cells of the genus Saccharomyces (Saccharomyces cerevisiae, Saccharomyces pombe, etc.), and molds, such as cells of the genus Aspergillus (Aspergillus niger), etc. The protein expression of) is well known and can be used as a host for the production of antibodies of the present invention.

In the case of using prokaryotic cells, there is a production system using bacterial cells. Bacterial cells other than the aforementioned E. coli are known to use a subtilis production system and can be used for the production of antibodies of the present invention.

In the case where the host cell of the present invention is used to generate an antibody, the host cell may be cultured with a expression vector containing a polynucleotide encoding the antibody of the present invention, and the polynucleotide may be expressed. Training According to a known method. For example, when animal cells are used as hosts, culture media can be used, such as: DMEM, MEM, RPMI1640, IMDM. In this case, serum rehydration such as FBS and fetal calf serum (FCS) may be used in combination, or cell culture may be performed in serum-free culture. The pH during culture is preferably about 6 to 8. The culture is usually carried out at about 30 to 40 ° C for about 15 to 200 hours, and the medium is added for exchange, aeration, and stirring as needed.

On the other hand, the system for producing polypeptides in vivo is, for example, an animal production system or a plant production system. The target polynucleotide is introduced into these animals or plants, and the polypeptide is produced and recovered in the animal or plant. The "host" in the present invention includes such animals and plants.

In the case of using animals, there are production systems using mammals and insects. As mammals, goats, pigs, sheep, mice, cattle, etc. can be used (Vicki Glaser, SPECTRUM Biotechnology Applications (1993)). When mammals are used, transgenic animals can be used.

For example, a polynucleotide encoding an antibody of the present invention and a gene encoding a polypeptide inherently produced in milk such as goat beta casein are modulated into a fusion gene. Next, a polynucleotide fragment containing the fusion gene is injected into a goat embryo, and the embryo is transferred to a female goat. The target antibody can be obtained from the milk produced by the transgenic goat or its offspring born from the embryo-contained goat. In order to increase the amount of milk containing antibodies produced by transgenic goats, appropriate hormones can also be administered to transgenic goats (Ebert et al., Bio / Technology (1994) 12: 699-702).

Moreover, the insect which produces the antibody of this invention can use silkworm, for example. When silkworms are used, baculoviruses inserted with a polynucleotide encoding the antibody of interest can be used to infect silkworms, and the antibodies of interest can be obtained from the silkworm body fluids (Susumu et al., Nature (1985) 315: 592-4).

Furthermore, in the case where a plant is used for the production of the antibody of the present invention, for example, tobacco can be used. When tobacco is used, a polynucleotide encoding an antibody of interest is inserted into a plant expression vector, such as pMON 530, and the vector is introduced into a bacterium of Agrobacterium tumefaciens. Infecting the bacteria with tobacco, such as Nicotiana tabacum, the desired antibodies can be obtained from the tobacco leaves (Ma et al., Eur. J. Immunol. (1994) 24: 131-8).

The antibody obtained in this way can be isolated from the inside or outside of the host cell (medium, milk, etc.), and purified into a substantially pure and uniform antibody. The isolation and purification of the antibody can be performed by any method generally used for purification of polypeptides, without any limitation. For example, chromatography columns, filtration membranes, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization can be appropriately selected. The antibodies are separated and purified in combination.

Examples of chromatography include: affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al. (1996) Cold Spring Harbor Laboratory Press). Such chromatography can be performed by liquid chromatography, for example, liquid chromatography such as HPLC or FPLC. Examples of affinity chromatography columns include: PROTEIN A column and PROTEIN G column. For example, a column using PROTEIN A includes Hyper D, POROS, Sepharose F.F. (manufactured by Pharmacia), and the like.

If necessary, some peptides can be arbitrarily modified or removed due to the action of an appropriate protein-modifying enzyme before or after purification of the antibody. protein As the qualitative modification enzyme, for example, trypsin, Chymotrpsin, lysine terminal peptidase, protein kinase, glucose oxidase, and the like can be used.

As described above, the method for producing a polypeptide variant or heteromultimer of the present invention including the steps of culturing the host cell of the present invention and recovering the polypeptide from the cell culture is also one of the preferred aspects of the present invention.

[Example]

Hereinafter, the present invention will be described in more detail with examples, but the present invention is not limited to these examples.

[Example 1] Preparation of non-neutralizing antibodies against Factor IXa (F.IXa)

1-1. Immune and fusion tumor production

Eight BALB / c mice (male, 6 weeks of age at the beginning of immunization, Charles‧River, Japan) and 5 MRL / lpr mice (male, 6 weeks of age at the beginning of immunization, Japanese Charles‧River) were divided as follows: Factor IXaβ (Enzyme Research Laboratories, Inc.) was immunized. For the first immunization, 40 μg / head was administered subcutaneously with FCA (Freud's complete adjuvant H37 Ra (Difco laboratories)) latexed Factor IXaβ. After 2 weeks, Factor IXaβ latexized with FIA (Difco laboratories) was administered subcutaneously at 40 μg / head. Thereafter, additional immunizations were performed 3 to 7 times at 1-week intervals. The serum antibody value of Factor IXaβ increased. After confirming with ELISA (Enzyme linked immunosorbent assay) shown in 1-2, the final immunization was diluted with PBS (-) (phosphate buffer solution without calcium and magnesium ions) to 40 μg. / head for intravenous administration. Three days after the final immunization, mouse spleen callus cells and mouse myeloma cells P3X63Ag8U.1 (referred to as P3U1, ATCC CRL-1597) were used PEG1500 (Roche Diagnostics) was used for cell fusion. Fusion cells suspended in RPMI1640 medium (Invitrogen) containing 10% FBS (Invitrogen) (hereinafter referred to as 10% FBS / RPMI1640) were inoculated into a 96-well culture plate and fused for 1, 2, 3, and 5 days, and then replaced by Select medium for HAT (10% FBS / RPMI1640 / 2% HAT 50x concentrate (Dai Nihon Pharmaceutical) / 5% BM-Condimed H1 (Roche Diagnostics)) for selective culture of fusion tumors. Using the culture supernatant taken on the 8th or 9th day after fusion, the binding activity to Factor IXa was determined by ELISA shown in 1-2, so as to select fusion tumors with Factor IXa binding activity. Next, the neutralizing activity against Factor IXa enzyme activity was measured by the method shown in 5-3, and fusion tumors were selected that did not have neutralizing activity against Factor IXa. The fusion tumor line was inoculated with 1 cell in one well of a 96-well culture plate, cloned by limiting dilution twice, and established as a fusion tumor XB12 that produced anti-Factor IXa antibodies.

1-2.Factor IXa ELISA

Factor IXaβ diluted to 1 μg / mL with coating buffer (100 mM sodium bicarbonate, pH 9.6, 0.02% sodium azide) was dispensed at 100 μL / well on Nunc-Immuno ^™ 96 MicroWell ^™ plates MaxiSorp ^(TM) (Nalge Nunc International)) and incubated overnight at 4 ° C. After washing 3 times with PBS (-) containing Tween ^(R) 20, it was diluted with dilution buffer (50 mM Tris-HCl, pH 8.1, 1% bovine serum albumin, 1 mM MgCl ₂ , 0.15 M NaCl , 0.05% Tween ^(R) 20, 0.02% sodium azide), and the plate was allowed to stand at room temperature for 2 hours for blocking. After removing the buffer, a culture supernatant of mouse antiserum or fusion tumor diluted with 100 μL / well of dilution buffer was added to the dish, and incubated at room temperature for 1 hour. After washing the plate 3 times, add 100 μL / well of alkaline phosphatase-labeled goat anti-mouse IgG (H + L) (Zymed Laboratories) diluted with dilution buffer to 1/2000, and incubate at room temperature for 1 hour. . After washing the plate 6 times, a coloring substrate Blue-Phos ^™ phosphate substrate (Kirkegaad & Perry Laboratories) 100 μL / well was added, and the plate was incubated at room temperature for 20 minutes. After adding 100 μL / well of Blue-Phos ^™ Stop Solution (Kirkegaad & Perry Laboratories), the absorbance at 595 nm was measured with a Microplate Reader Model 3550 (Bio-Rad Laboratories).

1-3. Determination of Factor IXa neutralizing activity

Phospholipid (Sigma-Aldrich) was dissolved in distilled water for injection and subjected to ultrasonic treatment to prepare a 400 μg / mL phospholipid solution. 40 μL of Tris buffered physiological saline solution (hereinafter referred to as TBSB) containing 0.1% bovine serum albumin and 10 μL of Factor IXaβ (Enzyme Research Laboratories) 10 μL and 5 μL of 400 μg / mL phospholipid solution and 100 μM CaCl ₂ , 20 mM MgCl ₂ 5 μL of TBSB and 10 μL of the fusion tumor culture supernatant were mixed in a 96-well plate and incubated for 1 hour at room temperature. 20 μL of 50 μg / mL Factor X (Enzyme Research Laboratories) and 10 μL of 3U / mL Factor VIIIa (Amrican diagnostica) were added to the mixed solution, and the mixture was reacted at room temperature for 30 minutes. The reaction was stopped by adding 10 μL of 0.5M EDTA to these. 50 μL of S-2222 solution (Chromogenix) was added to the reaction solution, and the mixture was incubated at room temperature for 30 minutes, and then measured with a Microplate Reader Model 3550 (Bio-Rad Laboratories, Inc.). The absorbance at a wavelength of 405 nm and a control wavelength of 655 nm was measured.

[Example 2] Production of non-neutralizing antibodies against Factor X (F.X)

2-1. Immune and fusion tumor production

BALB / c mice (male, 6 weeks of age at the start of immunization, Japanese Charles‧ 8 mice and 5 MRL / lpr mice (male, 6 weeks old at the beginning of immunization, Charles · River, Japan) were immunized with Factor X (Enzyme Research Laboratories) as follows. For the first immunization, Factor X40μg / head was injected subcutaneously with FCA latex. Two weeks later, FIA latexed Factor X 20 or 40 μg / head was administered subcutaneously. Thereafter, additional immunizations were performed every 1 week, and a total of 3 to 6 times were performed. After confirming that the antibody value of the antibody against Factor X increased by ELISA shown in 2-2, the final immunization was intravenously administered to Factor X 20 or 40 μg / head diluted in PBS (-). Three days after the final immunization, the mouse spleen callus cells and mouse myeloma cells P3U1 were subjected to cell fusion according to a conventional method using PEG1500. Fusion cells suspended in 10% FBS / RPMI1640 medium were inoculated into 96-well culture plates and replaced with HAT selection medium after fusion for 1, 2, 3, and 5 days to perform selective culture of fusion tumors. Using the culture supernatant taken on the 8th day after fusion, the binding activity to Factor X was measured according to the ELISA shown in 2-2. A fusion tumor with Factor X-binding activity was selected, and the neutralizing activity on the enzyme activity of Factor Xa was determined by the method shown in 2-3. Fusion tumors with no neutralizing activity to Factor Xa were cloned by limiting dilution twice to establish a fusion tumor SB04 that produces anti-Factor X antibodies.

2-2.Factor X ELISA

Factor X diluted to 1 μg / mL with coating buffer was dispensed at 100 μL / well on a Nunc-immunized plate, and then incubated at 4 ° C. overnight. After washing three times with Tween ^(R) 20 in PBS (-), the plate was blocked with dilution buffer at room temperature for 2 hours. After the buffer solution was removed, a mouse antiserum or a fusion supernatant of the fusion tumor diluted with the dilution buffer was added to the plate, and the plate was incubated at room temperature for 1 hour. After washing the disc 3 times, alkaline phosphatase-labeled goat anti-mouse IgG (H + L) diluted to 1/2000 with a dilution buffer was added and incubated at room temperature for 1 hour. After washing the dish 6 times, a coloring matrix Blue-Phos ^TM phosphate matrix (Kirkegaad & Perry Laboratories) 100 μL / well was added, and the plate was incubated at room temperature for 20 minutes. After adding 100 μL / well of Blue-Phos ^™ Stop Solution (Kirkegaad & Perry Laboratories), the absorbance at 595 nm was measured using a Microplate Reader Model 3550 (Bio-Rad Laboratories).

2-3. Determination of Factor Xa neutralizing activity

TBCP (2.78 mM CaCl ₂ , 22.2 μM phospholipids (phospholipids, choline: phospholipid filaments) containing fused tumor culture supernatants 10 μL and 40 μL of 250 pg / mL Factor Xa (Enzyme Research Laboratories) diluted to 1/5 with TBSB TBSB (amino acid = 75: 25, Sigma-Aldrich) was mixed and incubated at room temperature for 1 hour. To the mixed solution was added 20 μg / mL prothrombin (Enzyme Research Laboratories) and 100 ng / mL for activation. 50 μL of TBCP of Factor V (Factor Va (Haematologic Technologies)) was coagulated and reacted for 10 minutes at room temperature. 10 μL of 0.5 M EDTA was added to stop the reaction. 50 μL of 1 mM S-2238 solution (Chromogenix) was added to the reaction solution, After incubating at room temperature for 30 minutes, the absorbance at 405 nm was measured using a Microplate Reader Model 3550 (Bio-Rad Laboratories).

[Example 3] Construction of chimeric bispecific antibody expression vector

3-1. Self-fusion tumors modulate DNA fragments encoded as variable regions of antibodies

From fusion tumor XB12 producing anti-F.IXa antibody or fusion tumor SB04 producing anti-FX antibody, total RNA was extracted using QIAGEN ^(R) RNeasy ^(R) Mini Kit (QIAGEN) according to the method described in the instruction manual. Total RNA was dissolved in 40 μL of sterilized water. Using purified 1-2 μg of RNA as a template, a single-stranded cDNA was synthesized by RT-PCR using the SuperScript cDNA Synthesis System (Invitrogen) according to the method described in the instruction manual.

3-2. Amplification and sequence analysis of variable region of antibody H chain by PCR

For primers for amplification of the mouse antibody H chain variable region (VH) cDNA, a mixture of HB primers and a mixture of HF primers reported by Krebber et al. (J. Immunol. Methods 1997; 201: 35-55) were prepared. Using 0.5 μL of each 100 μM HB primer mixture and 100 μM HF primer mixture, prepare 25 μL of reaction solution (2.5 μl of cDNA solution prepared in 3-1, KOD plus buffer (Toyobo), 0.2 mM dNTPs, 1.5 mM MgCl ₂ , 0.75 unit DNA polymerase KOD plus (Toyobo)). The PCR uses a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer), which corresponds to the efficiency of cDNA fragment amplification. Condition A (after heating at 98 ° C for 3 minutes, 98 ° C for 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 30 seconds) The reaction was performed as 1 round for 32 rounds) or condition B (after heating at 94 ° C for 3 minutes, a reaction consisting of 94 ° C for 20 seconds, 46 ° C for 20 seconds, and 68 ° C for 30 seconds was taken as 1 round for 5 rounds, and then The reaction consisting of 94 ° C. for 20 seconds, 58 ° C. for 20 seconds, and 72 ° C. for 30 seconds was regarded as one round, and 30 rounds were performed). After PCR, the reaction solution was subjected to 1% agar gel electrophoresis. The amplified fragment of the desired size (about 400 bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μl of sterilized water. The base sequence of each DNA fragment was determined using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and a DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the method described in the attached specification. The sequence group determined by this method is compared and analyzed by analysis software GENETYX-SV / RC Version 6.1 (Genetyx), and those with different sequences are selected.

3-3. Modulation of DNA fragments in the variable region of an antibody for breeding

In order to attach the Sfi I cutting site for selection to both ends of the variable region amplified fragment of the antibody, the following operations were performed.

In order to amplify the Sfi I cut site plus the VH fragment (Sfi I-VH), prepare the primer (HBy (Sly) Ser) 2-linker sequence of the primer HB to change the sequence with the Sfi I cut site (primer VH-5 ' End). Using 0.5 μl of each 10 μM sequence-specific primer VH-5 ′ end and 10 μM primer scfor (J. Immunol. Methods 1997; 201: 35-55), 20 μL of the reaction solution (3-2 prepared purified VH cDNA amplified fragment solution) was prepared. 1 μl, KOD plus buffer (Toyobo), 0.2mM dNTPs, 1.5mM MgCl ₂ , 0.5 unit DNA polymerase KODplus (Toyobo)). For PCR, a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) was used. According to the efficiency of fragment amplification, the reaction was performed under condition A (heating at 98 ° C for 3 minutes, 98 ° C for 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 30 seconds. 32 rounds as one round) or condition B (after heating at 94 ° C for 3 minutes, a reaction consisting of 94 ° C for 20 seconds, 46 ° C for 20 seconds, and 68 ° C for 30 seconds is taken as 1 round for 5 rounds, and then at 94 ° C The reaction consisting of 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 30 seconds was performed as one round, and 30 rounds were performed). After PCR, the reaction solution was subjected to 1% agar gel electrophoresis. The amplified fragment of the target size (about 400 bp) was purified using a QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μL of sterilized water.

In order to amplify the mouse antibody L chain variable region (VL) cDNA fragment, first prepare 0.5 μL of each 100 μM LB primer mix and 100 μM LF primer described in the report of Krebber et al. (J. Immunol. Methods 1997; 201: 35-55) 25 μL of the reaction mixture (2.5 μL of c-DNA solution prepared by 3-1, KOD plus buffer (Toyobo), 0.2mM dNTPs, 1.5mM MgCl ₂ , 0.75 unit DNA polymerase KOD plus (Toyobo)) . The PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer). According to the efficiency of the fragment amplification, the following conditions were used: 94 ° C for 3 minutes, 94 ° C for 20 seconds, 46 ° C for 20 seconds, and 68 ° C for 30 seconds. The reaction was performed as 1 round, and 5 rounds were performed, and a reaction consisting of 94 ° C for 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 30 seconds was taken as 1 round for 30 rounds. After PCR, the reaction solution was subjected to 1% agar gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using a QIAqucick Gel Extractio kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μL of sterilized water. This fragment is a state obtained by adding a primer (LF) from the LF (Gly4Ser) 3-linker sequence to the C-terminus. In order to add an Sfi I cut site to the C-terminus of this fragment, it was prepared to change the (Gly4Ser) 3-linker sequence of the primer LF to have the sequence shown in the Sfi I cut site (primer VL-3 'end). In order to amplify the Sfi I cut site addition VL fragment (Sfi I-VL), use 0.5 μL of each 10 μM VL-3 ′ terminal primer mixture and 10 μM scback primer to prepare 20 μL of reaction solution (1 μL of purified VL cDNA amplified fragment solution, KOD plus buffer (Toyobo), 0.2mM dNTPs, 1.5mM MgCl ₂ , 0.5 unit DNA polymerase KOD plus (Toyobo)). The PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) under the following conditions: After heating at 94 ° C for 3 minutes, a reaction consisting of 94 ° C for 20 seconds, 46 ° C for 20 seconds, and 68 ° C for 30 seconds was performed as a round, and 5 cycles were performed. For the round, a reaction consisting of 94 ° C for 20 seconds, 58 ° C for 20 seconds, and 72 ° C for 30 seconds was taken as one round, and 30 rounds were performed. . After PCR, the reaction solution was subjected to 1% agar gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μL of sterilized water.

The purified Sfi I-VH and Sfi I-VL fragments were prepared by Sfi I (Takarabio) according to the method described in the attached instruction manual, and digested overnight at 50 ° C. Thereafter, the reaction solution was purified using a QIAquick PCR Purification Kit (QIAGEN) according to the method described in the attached instruction manual, and was dissolved in 30 μL of the buffer solution EB attached to the kit.

3-4. Human IgG4-mouse chimeric bispecific IgG antibody expression plastids

In order to generate a bispecific IgG antibody of interest, in order to form hetero molecules of each H chain, refer to the knobs-into-holes technology of IgG1 (Non-Patent Document 3) to produce an amino acid substitute for the CH3 portion of IgG4 . Type a (IgG4γa) is a substitute for Y349C and T366W, and type b (IgG4γb) is a substitute for E356C, T366S, L368A, and Y407V. Furthermore, substitutions (-ppcpScp->-ppcpPcp-) were also introduced in the hinge region of the two substituents. According to the present technology, although most heterogeneous bodies are obtained, the L chain is not limited to this, and the generation of unnecessary antibody molecules may affect the subsequent activity measurement. Therefore, in order to efficiently produce a target-specific bispecific IgG antibody in a cell that expresses one arm (called an HL molecule) of each specific antibody molecule separately, this method is an expression carrier corresponding to each HL molecule. , Is induced by the use of different agents.

For the expression of the single arm of the antibody molecule (referred to as the right arm HL molecule for short), the tetracycline-inducible vector pcDNA4 (Invitrogen) was inserted to embed the H chain to L chain regions, that is, used in animal cells A suitable mouse antibody variable region (VH to VL) and a human IgG4γa constant region are embedded downstream of the signal sequence (IL3ss) (Proc. Natl. Acad. Sci. USA. 1984; 81: 1075) Domain (sequence number: 9) to the κ constant region (sequence number: 10) (pcDNA4-g4H to pcDNA4-g4L). First, pcDNA4 was digested with Eco RV and Not I (Takarabio), a restriction enzyme cleaving site present in its multiple selection site. Chimeric bispecific antibodies with appropriate antibody variable regions of the right arm H chain to L chain expression units (about 1.6kb to about 1.0kb each) were digested with Xho I (Takarabio), and then QIAquick PCR Purification Kit (QIAGEN ) Purified in accordance with the method described in the attached specification, and reacted at 72 ° C. for 10 minutes using the DNA polymerase KOD (Toyobo) according to the composition of the reaction solution described in the attached instruction to smooth the ends. This smoothed-end fragment was purified by the QIAquick PCR Purification Kit (QIAGEN) according to the method described in the attached instruction manual, and digested with Not I (Takarabio). The Not I-blunt-end fragments (approximately 1.6 kb to 1.0 kb each) and pcDNA4 digested with the Eco RV-Not I were subjected to a ligation reaction using Ligation High (Toyobo) according to the method described in the attached specification. The reaction solution was used to transform the E. coli DH5α strain (Competent high DH5α (Toyobo)) into shape. The obtained ampicillin-resistant colonies were isolated from each plastid DNA using the QIAprep Spin Miniprep Kit (QIAGEN).

For the other single arm (referred to simply as the left arm HL molecule), the H chain was inserted into the region of the L chain in the ecdysone analog-inducible vector pIND (Invitrogen) according to the method described above, that is, for animal cells. A suitable mouse antibody variable region (VH to VL) and a human IgG4γb invariant region (sequence number: 11) to a κ invariant region are embedded downstream of the signal sequence (IL3ss) (EMBO.J.1987; 6: 2939). (PIND-g4H to pIND-g4L) and isolate each plastid DNA.

3-5. Construction of a bispecific antibody expression vector

Tetracycline-inducible plastids (pcDNA4-g4H to 3-4 modulation) pcDNA4-g4L) was digested with Sfi I, and the reaction solution was subjected to 1% agar gel electrophoresis. A fragment (approximately 5 kb) from which the variable region portion (VH to VL) of the original antibody was removed was purified by a QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and was dissolved in 30 μL of sterilized water. This fragment and Sfi I-digested anti-F.IXa antibody XB12 derived from 3-3 modulation were derived from Sfi I-VH to Sfi I-VL fragments in the Quick Ligation Kit (New England Biolabs) according to the method described in the attached instruction sheet. Perform a linking reaction. The reaction solution was used to transform the coliform DH5α strain (Competent high DH5α (Toyobo)). In addition, Sfi I prepared from 3-4 digested ecdysone-like inducible expression plastids (pIND-g4H to pIND-g4L), and the fragments of the variable region portion (VH to VL) of the antibody were removed in the same manner as above. Sfi I-VH to Sfi I-VL fragments derived from Sfi I-digested anti-FX antibody SB04 prepared by the corresponding 3-3 were embedded in the same way.

The base sequence of each DNA fragment was determined using a BigDye Terminator cycle sequencing kit (Applied Biosystems), and a DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the method described in the attached specification. The sequence group determined by this method is analyzed by the analysis software GENETYX-SV / RC Version 6.1 (Genetyx).

From this colony, the plastid DNA was isolated using a QIAprep Spin Miniprep kit (QIAGEN) and dissolved in 100 μL of sterilized water. Anti-F.IXa antibody chimeric H chain expression vector, anti-F.IXa antibody chimeric L chain expression vector, anti-FX antibody chimeric H chain expression vector, and anti-FX antibody chimeric L chain expression vector were named pcDNA4-g4 XB12H , PcDNA4-g4 XB12L, pIND-g4 SB04H and pIND-g4 SB04L.

[Example 4] Preparation of chimeric bispecific antibodies

4-1. Preparation of DNA solution

The HL molecule expression vectors for the right arm of the antibody (pcDNA4-g4 XB12H and pcDNA4-g4 XB12L) were induced with tetracycline. In order to completely suppress the expression in the absence of tetracycline, plastid pcDNA6 / TR (Invitrogen) encoding a Tet repressor is required. The vectors for expressing the HL molecule of the left arm of the antibody (pIND-g4 SB04H and pIND-g4 SB04L) were induced by the expression of insect hormone ecdysone analog (ponasterone A). At this time, ecdysone receptors and retinol X receptors that are coded to react with and induce ponasterone A are required. PVgRXR (Invitrogen). Therefore, a total of six plastid DNA mixed solutions for transfecting animal cells were prepared. To prepare 10 mL of cell culture fluid, pcDNA4-g4 XB12H, pcDNA4-g4 XB12L, pIND-g4 SB04H and pIND-g4 SB04L were each 3 μg, and pcDNA6 / TR and pVgRXR were each 18 μg.

4-2. Transfection of animal cells

HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% FCS (MOREGATE), and inoculated to a culture dish for adherent cells at a cell density of 5 × 10 ⁵ cells / mL (10 cm in diameter, CORNING) 10 mL of each dish, placed in a CO ₂ incubator (37 ° C, 5% CO ₂ ) and cultured for one day and night. Add the 4-1 prepared plastid DNA mixture to the transfection reagent, 75.8 μL of Lipofectamine 2000 (Invitrogen) and 2708 μL of Opti-MEM I medium (Invitrogen), and leave it at room temperature for 20 minutes and put it into each well. The cells were cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for 4 to 5 hours.

4-3. Induction of expression of bispecific IgG antibodies

Aspirate the medium from the transfected cell culture solution from the previous method, and put 10 mL of CHO-S-SFM-II (Invitrogen) medium containing 1 μg / mL tetracycline (Wako Pure Chemical Industries) into a CO ₂ incubator. (37 ° C, 5% CO ₂ ) was cultured for 1 day, and the first expression induction of the HL molecule in the right arm of the antibody was performed. After that, the culture medium was removed by suction, and then washed with 10 mL of CHO-S-SFM-II medium, and then 10 mL of CHO-S-SFM-II medium containing 5 μM of ponasterone A (Invitrogen) was put in a CO ₂ incubator (37 ° C, 5% CO ₂ ) was cultured for 3 days, and the second expression induction of the HL molecule of the left arm of the antibody was performed to secrete the bispecific IgG antibody into the culture medium. After the culture supernatant was recovered, the cells were removed by centrifugation (about 2000 g, 5 minutes, room temperature), and sterilized by passing through a 0.22 μm filter MILLEX ^(R) -GV (Millipore). The sample was stored at 4 ° C until use.

4-4. Antibody purification

100 μL rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences) was added to 10 mL of the culture supernatant obtained in the method of Example 4-3, and the mixture was inverted and mixed at 4 ° C. for more than 4 hours. The solution was transferred to a 0.22 μm filter cup Ultrafree ^(R) -MC (Millipore), and washed three times with 500 μL of TBS containing 0.01% Tween ^(R) 20, and then the rProtein A Sepharose ^TM resin was suspended in 0.01% containing 100 μL Tween ^(R) 20 10 mM HCl, pH 2.0, and allowed to stand for 2 minutes to elute the antibody. Immediately add 5 μL of 1M Tris-HCl, pH 8.0 to neutralize.

4-5. Quantification of human IgG concentration

Goat anti-human IgG (Biosource International) was prepared to 1 μg / mL with a coating buffer, and immobilized on a Nunc-immunity plate (Nunc). After blocking treatment with dilution buffer (DB), a culture supernatant sample appropriately diluted with DB is added. In addition, the standard used to calculate the antibody concentration was similarly added to human IgG4 at 11 stages in a DB series diluted three times from 2000 ng / mL (human-type anti-TF antibody, see WO 99/51743). After 3 washes, goat anti-human IgG and alkaline phosphatase (Biosource International) were reacted. After 5 washes, coloring was performed using Sigma 104 ^(R) phosphorolytic enzyme matrix (Sigma-Aldrich) as a matrix, and absorbance was measured at a reference wavelength of 655 nm and 405 nm using an absorbance reader Model 3550 (Bio-Rad Laboratories). Microplate Manager III (Bio-Rad Laboretories) software was used to calculate the human IgG concentration in the culture supernatant from the standard calibration curve.

[Example 5] Analysis of plasma coagulation

To determine whether it is a bispecific antibody that is effective for the blood coagulation ability of hemophilia A, the effect of this antibody on the thromboxin time (APTT) of the activated fraction of Factor VIII-deficient plasma was investigated. A mixture of 50 μL of antibody solution of various concentrations, 50 μL of Factor VIII-deficient plasma (Biomerieux), and 50 μL of APTT reagent (Dade Behring) was heated at 37 ° C. for 3 minutes. The coagulation reaction was started by adding 50 μL of 20 mM CaCl ₂ (Dade Behring) to the mixture. The time from the connection of KC10A (Amelung) of CR-A (Amelung) to the coagulation was measured.

Using a calibration curve made when the clotting time of Factor VIII-deficient plasma was set to 0% and the clotting time of normal plasma was set to 100%, the similar activity of the factor VIII of the bispecific antibody was calculated from the coagulation time of the addition of the bispecific antibody (% ).

[Example 6] Humanization of bispecific antibodies

The anti-FactorIXa antibody XB12 and the anti-FactorX antibody SB04, which have the fastest reduction in blood clotting time, are humanized as follows.

6-1. Identity search of human antibodies

Obtain human antibody amino acids from the generally published Kabat database (ftp: //ftp.ebi.ac.uk/pub/databases/kabat/) and IMGT database (http://imgt.cines.fr/) Sequence data and use the constructed database to divide into mouse XB12-H chain variable region, mouse XB12-L chain variable region, mouse SB04-H chain variable region, and mouse SB04-L chain variable region Identity Retrieval. As a result, it was confirmed that they have high identity with the human antibody sequences shown below, and therefore were used as a framework region of humanized antibodies (hereinafter referred to as FR).

(1) XB12-H chain variable region: KABATID-020619 (Kabat database)

(Mariette et al., Arthritis Rheum. 1993; 36: 1315-1324)

(2) XB12-L chain variable region: EMBL Accession No. X61642 (IMGT database)

(Mark et al., J Mol Biol. 1991; 222: 581-597.)

(3) SB04-H chain variable region: KABATID-025255 (Kabat database)

(Demaison et al., Immunogetetics 1995; 42: 342-352)

(4) SB04-L chain variable region: EMBL Accession No. AB064111 (IMGT database)

(Unpublished information)

Humanized antibodies in which the complementary epitope regions (hereinafter referred to as CDRs) of the respective mouse antibodies were transplanted into the human antibody FRs (1) to (4) were prepared.

In addition, using the identity search website (http://www.ncbi.nlm.nih.gov/BLAST/) generally published by the NCBI to search for human antibody secretions with high identity to (1)-(4) human antibodies Signal sequence. Using the search, the secretion signal sequence shown below was obtained.

(1) XB12-H chain variable region: GenBank Accession No. AF062120

(2) XB12-L chain variable region: GenBank Accession No. M74019

(3) SB04-H chain variable region: GenBank Accession No. BC019337

(4) SB04-L chain variable region: GenBank Accession No. AY204756

6-2. Construction of humanized antibody gene expression vector

From the base sequence encoding the amino acid sequence of the autocrine signal sequence to the variable region of the antibody, twelve synthetic oligo DNAs of about 50 bases with 3 bases of about 20 bases are alternately made. Furthermore, a primer is attached to the 5 'end of the antibody variable region gene and has a XhoI cut sequence, and a primer is attached to the 3' end of the antibody variable region gene and has a SfiI cut sequence.

The synthetic oligo DNA prepared into 2.5 μM was mixed with 1 μL each, and 1 × TaKaRa Ex Taq buffer, 0.4 mM dNTPs, 0.5 units of TaKaRa Ex Taq (both manufactured by Takara Shuzo) were added to prepare 48 μL of the reaction solution. After 5 minutes of incubation at 94 ° C, a reaction consisting of 94 ° C for 2 minutes, 55 ° C for 2 minutes, and 72 ° C for 2 minutes was performed for 2 rounds, and a combination of each synthetic oligo DNA and an elongation reaction were performed. Next, 1 μL of primers (10 μM each) adhered to the 5 ′ end and the 3 ′ end of the antibody gene were added, and a reaction consisting of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute was performed for 35 rounds, and the reaction was performed at 72 ° C for 5 minutes. Minutes to amplify the antibody variable region genes. After PCR, the entire amount of the reaction solution was subjected to 1% agar gel electrophoresis. The amplified fragment of the desired size (about 400 bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μl of sterilized water. This fragment was cloned using pGEM-T Easy Vector Systems (Promega) according to the method described in the attached instruction sheet. The base sequence of each DNA fragment was applied using the BigDye Terminator cycle sequencing kit (Applied Biosystems), and the DNA sequencer ABI PRISM 3700 DNA Sequencer (Applied Biosystems) were sequenced according to the methods described in the attached instructions.

After the plastids confirmed as the correct humanized antibody variable region gene sequence were digested with EcoRI and SfiI, the reaction solution was subjected to 1% agarose gel electrophoresis. A DNA fragment of the desired size (about 400 bp) was purified using a QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μl of sterilized water. In addition, the tetracycline-inducible plastids (pcDNA4-g4H, pcDNA4-g4L) and ecdysone analog-inducible plastids (pIND-g4H, pIND-g4L) prepared in Example 3-3 were prepared using EcoRI and After SfiI digestion, a fragment (about 5 kb) containing an antibody-invariant region was purified using a QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μl of sterilized water. Humanized XB12 antibody gene fragment (H chain variable region or L chain variable region) digested with EcoRI and SfiI and tetracycline-inducible expression plastids (pcDNA4-g4H, pcDNA4-g4L) digested with EcoRI and SfiI ), A Rapid DNA Ligation Kit (Roche Diagnostics) was used to perform the ligation reaction according to the method described in the attached instruction sheet. In addition, humanized SB04 antibody gene fragments (H chain variable region or L chain variable region) digested with EcoRI and SfiI and ecdysone analog-inducible expression plastids (pIND-g4H, pIND -g4L) The Rapid DNA Ligation Kit (Roche Diagnostics) was used to perform the ligation reaction according to the method described in the attached instruction sheet. Shape conversion of E. coli DH5α strain (Toyobo) was performed using a part of each reaction solution.

In addition, in order to express a non-bispecific antibody as a normal humanized antibody, a expression vector was prepared as follows. PCAGGS (Niwa et al. 1991 Gene, 108: 193-199.) With chicken beta actin promoter inserted into plastids (pCAG-g4H, pCAG-gκ) with invariant regions of wild-type antibodies To XhoI and SfiI digestion, and a humanized XB12 antibody gene fragment (H chain variable region or L chain variable region) or humanized SB04 antibody gene fragment prepared by inserting the above-mentioned bispecific antibody expression vector and digested with XhoI and SfiI was inserted (H-chain variable region or L-chain variable region). For the DNA ligation reaction, the Rapid DNA Ligation Kit (Roche Diagnostics) was used to transform the coliform DH5α strain (Toyobo).

6-3. Modulation of humanized bispecific antibodies

Using four humanized bispecific antibody expression vectors and pcDNA6 / TR, pVgRXR, gene introduction and expression induction of HEK293H were performed by the methods shown in Examples 4-2, 4-3. Furthermore, antibody purification and antibody concentration were quantified by the methods shown in Examples 4-5 and 4-6.

6-4. Modulation of humanized antibodies

In order to express the normal humanized antibody of a non-bispecific antibody, the humanized H chain antibody expression vector and the humanized L chain antibody expression vector prepared in Example 6-3 were used to gene HEK293H according to the method shown in Example 4-2. Import. After gene introduction, 10 mL of CHO-S-SFM-II medium (Invitrogen) was added, and after removing and washing, 10 mL of CHO-S-SFM-II was added and placed in a CO ₂ incubator (37 ° C, 5% CO ₂ ). After 3 days of culture, humanized antibodies were secreted.

6-5. Evaluation of the activity of humanized bispecific antibodies and changes in antibody sequences

In order to evaluate the plasma coagulation ability of the prepared humanized bispecific antibody and chimeric bispecific antibody (XB12 / SB04), the effect of antibody on APTT was investigated using F.VIII-deficient plasma according to the method of Example 5. For humanized bispecific antibodies with low blood coagulation ability, aim at increasing activity to change human resistance The amino acid of the body FR. Furthermore, the cysteine residue of CDR3 of the XB12 antibody VH, which is concerned about low thermal stability, was also changed to alanine residue. Specifically, a mutation was introduced into the variable region of a humanized antibody using the QuikChange point mutation kit (Stratagene) according to the method described in the attached specification. By repeatedly evaluating the amino acid changes and blood coagulation ability of the FR sequence, a humanized bispecific antibody (humanized XB12 antibody (VH: hXB12f-A, VL: hXBVL)) having the same activity as XB12 / SB04 was obtained / Humanized SB04 antibody (VH: hSB04e, VL: hSBVL-F3f). The sequence of the variable region of each antibody is represented by the following sequence number.

(1) Humanized XB12 antibody VH (hXB12f-A) sequence number: 1 (base sequence), sequence number: 2 (amino acid sequence)

(2) Humanized XB12 antibody VL (hXBVL) sequence number: 3 (base sequence), sequence number: 4 (amino acid sequence)

(3) Humanized SB04 antibody VH (hSB04e) sequence number: 5 (base sequence), sequence number: 6 (amino acid sequence)

(4) Humanized SB04 antibody VL (hSBVL-F3f) sequence number: 7 (base sequence), sequence number: 8 (amino acid sequence)

[Example 7] Modeling of humanized antibodies

In order to confirm the amino acid residues at the interface between VH and VL of humanized SB04 antibody, MOE software (Chemical Computing Group Inc.) was used to make an antibody Fv region model with the same degree simulator. In the interface between VH and VL, the amino acids of H39 and L38 are both glutamine (Gln), and it is confirmed that the side chains of the two residues form hydrogen bonds (Fig. 1 (A)). In addition, the amino acids of H45 and L44 were leucine (Leu) and proline (Pro), and it was confirmed that the side chains of the two residues were very close and formed a hydrophobic core (Fig. 1 (B)). These 2-position amino acid residues have been reported to be high in human antibodies Conservative (Vargas-Madrazo E et al. J. Mol. Recognit. 2003, 16: 113-120). For the numbers of H39, L38, H45, L44 and other antibodies, refer to the literature of Kabat et al. (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH).

[Example 8] Preparation and evaluation of modified humanized antibodies of amino acids of H39 and L38

8-1. Construction of modified antibody expression vectors for H39 and L38

By inhibiting the assembly of the humanized XB12 H chain and the humanized SB04 L chain, the glutamine in humanized XB12 H chain and the glutamine in L38 of humanized SB04 L chain were replaced in accordance with the findings of Example 7. . Specifically, the two amino acids (H39, L38) are replaced with a positively charged lysine (Lys) or arginine (Arg ), Glutamic acid (Glu) or aspartic acid (Asp) with negatively charged side chains. The humanized antibody gene was replaced with a QuikChange point mutation kit (Stratagene) according to the method described in the attached instruction sheet to introduce mutations. Each amino acid-substituted humanized antibody gene fragment was inserted into the bispecific antibody expression vector or the usual antibody expression vector used in Example 6-2.

8-2. Preparation of antibodies for assembly control evaluation and evaluation of assembly control

In order to evaluate the assembly control of H chain and L chain, three kinds of antibodies including humanized XB12 H chain (H39 change), humanized SB04 L chain (L38 change), and wild type humanized XB12 L chain were used as expression vectors. The method shown in Example 4-2 was used to introduce genes into HEK293H to secrete antibodies in the culture supernatant. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

200 ng of purified antibody was reduced in a sample buffer (TEFCO), injected into a 14% SDS-PAGE mini gel (TEFCO), and electrophoresed. After electrophoresis, immerse in a 7% acetic acid solution containing 10% methanol for 30 minutes for fixation, and immerse in SYPRO ^(R) Ruby protein gel staining solution (BIO-RAD) for day and night for staining. Next, immerse in a 7% acetic acid solution containing 10% methanol for 1 hour for decolorization, and use a fluorescence detection device FluorImagerSI (Amersham Biosciences) to perform image analysis to obtain an image. Using the obtained images, the fluorescence intensities of the H-chain and L-chain bands were calculated using ImageQuant ver4.2 (Amersham Biosciences).

The results are shown in Figure 2. Using the calculated fluorescence intensity intensity value, the ratio (%) of the target XB12-L chain is calculated as "XB12-L chain / L chain total (XB12-L chain + SB04-L chain) x 100". It was confirmed that the case where the amino acid of the humanized XB12 H chain (H39) and the humanized SB04 L chain (L38) was wild-type glutamine (Gln) was 50%, compared to the case where the H39 and L38 were replaced In the case of humanized XB12 L chain, the ratio of glutamic acid (Glu) was 82%, a 1.6-fold increase.

8-3. Preparation of bispecific antibodies for evaluation of coagulation activity and evaluation of coagulation activity

In order to evaluate the coagulation activity, the humanized XB12 H chain (H39 change) and humanized SB04 L chain (L38 change) bispecific antibody expression vector and wild type humanized XB12 L chain and humanized SB04 H chain were used. The antibody expression vector, pcDNA6 / TR, and pVgRXR were introduced into HEK293H by the method shown in Examples 4-2, 4-3, and expression induction was performed. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

Evaluation of the coagulation activity was performed by the method shown in Example 5. The results are shown in Figure 3. Show. Among the glutamate (Glu: E) -modified antibodies whose proportion increased to 82% in the evaluation of assembly control, it was confirmed that they exhibited the same or more coagulation activity as compared with the wild type.

8-4. Preparation of antibodies for binding activity evaluation

In order to evaluate the binding activity with FactorIXa and FactorX, humanized XB12H chain (modified H39) and wild-type humanized XB12 L chain antibody expression vectors or wild-type humanized SB04 H chain and humanized SB04 L chain (modified L38) antibody were used The expression vector was introduced into HEK293H by the method shown in Example 4-2, and the antibody was secreted into the culture supernatant. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

The evaluation of the binding activity of FactorIXa and FactorX was performed by the methods shown in Examples 1-2 and 2-2. The results are shown in Figures 4 and 5. It was confirmed that the binding activity did not change even when the amino acids of H39 and L38 were substituted.

From the above results, it is suggested that by changing the H39 of the XB12 H chain and L38 of the SB04 L chain, the biological activity such as the binding activity to the antigen and the coagulation activity instead of FactoVIII can be reduced, and the ratio of the target bispecific antibody can be made. rise. For example, the introduction of amino acid mutations at only one position in a polypeptide allows the assembly to be controlled without degradation. The method including protrusions and voids has not been reported to this extent, and it can be called the first insight.

[Example 9] Preparation and evaluation of modified humanized antibody of amino acid of L44

9-1. Construction of L44 modified antibody expression vector

By inhibiting the assembly of the humanized XB12 H chain and the humanized SB04 L chain, the proline acid of the humanized SB04 L chain L44 was replaced with a side chain charged amino acid according to the findings of Example 7. Specifically, the VH and VL boundaries Proline acid with a hydrophobic core on the surface is replaced with lysine (Arg) or arginine (Arg) with a positive charge in the side chain, glutamine (Glu) or aspartic acid (Glu) with a negative charge in the side chain Asp). The humanized antibody gene was replaced with a QuikChange point mutation kit (Stratagene), and mutations were introduced according to the method described in the attached specification. The amino acid-substituted humanized antibody gene fragment was inserted into the bispecific antibody expression vector or the usual antibody expression vector used in Example 6-2.

9-2. Preparation of antibodies for assembly control evaluation and evaluation of assembly control

In order to evaluate the assembly control of the H chain and the L chain, three kinds of antibody expression vectors such as humanized SB04 L chain (L44 change), wild-type humanized XB12 H chain, and wild-type humanized XB12 L chain were used. Example 4 was used. The method shown in -2 was used to introduce genes into HEK293H to secrete antibodies into the culture supernatant. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

200 ng of purified antibody was reduced in a sample buffer (TEFCO), injected into a 14% SDS-PAGE mini gel (TEFCO), and electrophoresed. After electrophoresis, immerse in a 7% acetic acid solution containing 10% methanol for 30 minutes for fixation, and immerse in SYPRO ^(R) Ruby protein gel staining solution (BIO-RAD) for day and night for staining. Next, immerse in a 7% acetic acid solution containing 10% methanol for 1 hour for decolorization, and use a fluorescence detection device FluorImagerSI (Amersham Biosciences) to perform image analysis to obtain an image. Using the obtained images, the fluorescence intensities of the H-chain and L-chain bands were calculated using ImageQuant ver4.2 (Amersham Biosciences).

The results are shown in Figure 6. Using the calculated fluorescence intensity intensity value, use the "XB12-L chain / L chain total (XB12-L chain + SB04-L chain) x 100" to calculate the proportion (%) of the target XB12-L chain. Confirmation: Humanized SB04 L-chain (L44) In the case where the amino acid is wild-type proline (Pro), 47%, in contrast, in the case of replacing L44, the proportion of the humanized XB12 L chain increased by 86-90%, which increased by 1.8-1.9 times.

9-3. Preparation of Bispecific Antibodies for Evaluation of Coagulation Activity and Evaluation of Coagulation Activity

To evaluate the coagulation activity, the humanized SB04 L chain (L44 altered) bispecific antibody expression vector and the wild-type humanized XB12 H chain, humanized XB12 L chain, and humanized SB04 H chain bispecific antibody expression vector were used. , PcDNA6 / TR, and pVgRXR, gene introduction and expression induction of HEK293H were performed by the methods shown in Examples 4-2, 4-3. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

Evaluation of the coagulation activity was performed by the method shown in Example 5. The results are shown in Figure 7. It was confirmed that among all the modified antibodies whose proportion was increased in the evaluation of assembly control, the coagulation activity was higher than that of the wild type.

9-4. Preparation of antibodies for binding activity evaluation

In order to evaluate the binding activity to FactorX, a wild-type humanized SB04 H chain and a humanized SB04 L chain (L44 altered) antibody expression vector were used to introduce the gene into HEK293H by the method shown in Example 4-2, so that the antibody was secreted in culture. Supernatant. In addition, the antibody concentration in the culture supernatant was quantified by the method shown in Example 4-6.

Evaluation of the binding activity of FactorX was performed using the culture supernatant by the method shown in Example 2-2. The results are shown in Figure 8. It was confirmed that the binding activity did not change even if the amino acid of L44 was substituted.

From the above results, it is implied that by placing L44 of the SB04 L chain in a position The change of amino acid can increase the proportion of the target bispecific antibody without reducing the biological activity such as the binding activity to the antigen and replacing the coagulation activity of FactoVIII. An example in which amino acid mutation is introduced only at one position in the polypeptide can control the assembly without inferior function. The method including protrusions and voids has not been reported to this extent, and it can be called the first.

[Example 10] Preparation and evaluation of modified humanized antibodies of amino acids of H39 and L38 and amino acids of L44

10-1. Construction of modified antibody expression vectors for H39, L38 amino acids and L44

By inhibiting the assembly of the humanized XB12 H chain and the humanized SB04 L chain, the humanized XB12 H chain and the humanized SB04 L chain L38 and L44 were replaced with side chain bands according to the findings of Examples 8 and 9. Charged amino acid. Specifically, H39 of the humanized XB12 H chain and L38 of the humanized SB04 L chain were replaced with the most effective glutamic acid (Glu) in Example 8, and will be present in the humanized SB04 L The proline acid of L44 of the chain is replaced with lysine (Arg) or arginine (Arg) with a positive charge on the side chain, glutamine (Glu) or aspartic acid (Asp) with a negative charge on the side chain . The humanized antibody gene was replaced with a QuikChange point mutation kit (Stratagene), and mutations were introduced according to the method described in the attached specification. The amino acid-substituted humanized antibody gene fragment was inserted into the bispecific antibody expression vector or the usual antibody expression vector used in Example 6-2.

10-2. Preparation of antibodies for assembly control evaluation and evaluation of assembly control

In order to evaluate the assembly control of H chain and L chain, three types of humanized SB04 L chain, humanized XB12 H chain and wild type XB12 L chain were used. The antibody expression vector was introduced into HEK293H by the method shown in Example 4-2, and the antibody was secreted in the culture supernatant. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

200 ng of purified antibody was reduced in a sample buffer (TEFCO), injected into a 14% SDS-PAGE mini gel (TEFCO), and electrophoresed. After electrophoresis, immerse in a 7% acetic acid solution containing 10% methanol for 30 minutes for fixation, and immerse in SYPRO ^(R) Ruby protein gel staining solution (BIO-RAD) for day and night for staining. Next, immerse in a 7% acetic acid solution containing 10% methanol for 1 hour for decolorization, and use a fluorescence detection device FluorImagerSI (Amersham Biosciences) to perform image analysis to obtain an image. Using the obtained images, the fluorescence intensities of the H-chain and L-chain bands were calculated using ImageQuant ver4.2 (Amersham Biosciences).

The results are shown in Figure 9. Using the calculated fluorescence intensity intensity value, the ratio (%) of the target XB12-L chain is calculated as "XB12-L chain / L chain total (XB12-L chain + SB04-L chain) x 100". It was confirmed that the diamino acids of humanized XB12 H chain (H39) and humanized SB04 L chain (L38) were changed to glutamic acid (Glu), and the humanized SB04 L chain (L44) was wild-type proline ( Pro) is 82%, the ratio of humanized XB12 L chain to humanized XB12 H chain (H39) and humanized SB04 L chain (L38) is changed to glutamic acid (Glu) and replaced by L44. It rose to 94-96%. The increase of this ratio is higher than 86-90% of L44 alone in Example 9.

10-3. Preparation of bispecific antibodies for evaluation of coagulation activity and evaluation of coagulation activity

In order to evaluate the coagulation activity, a modified humanized XB12 H chain was used, Humanized XB12 L chain and humanized SB04 H chain bispecific antibody expression vector and wild type humanized XB12 H chain, humanized XB12 L chain and humanized SB04 H chain bispecific antibody expression vector, pcDNA6 / TR, pVgRXR, Gene introduction and expression induction of HEK293H were performed by the methods shown in Examples 4-2 and 4-3. Furthermore, antibody purification and antibody concentration quantification were performed by the methods shown in Examples 4-5 and 4-6.

Evaluation of the coagulation activity was performed according to the method shown in Example 5. The results are shown in Figure 10. Among all the modified antibodies whose proportion was increased in the evaluation of assembly control, it was confirmed that the coagulation activity was equivalent to that of the wild type.

10-4. Preparation of antibodies for binding activity evaluation

In order to evaluate the binding activity to FactorX, using wild-type humanized SB04 H chain and variant humanized SB04 L-chain antibody expression vectors, HEK293H gene was introduced according to the method shown in Example 4-2, and the antibody was secreted in the culture supernatant. in. In addition, the antibody concentration in the culture supernatant was quantified by the method shown in Example 4-6.

Evaluation of the binding activity of FactorX was performed using the culture supernatant by the method shown in Example 2-2. The results are shown in Figure 11. It was confirmed that even when the L38 and L44 diamino acids were substituted, the binding activity did not change.

From the above results, it is suggested that by changing the amino acids of H39 of the XB12 H chain and L38 and L44 of the SB04 L chain, the biological activity such as binding activity to the antigen and coagulation activity instead of Facto VIII can be reduced, and the purpose is dual specificity. The proportion of antibodies has increased. It was confirmed that the proportion of bispecific antibodies was increased by increasing the number of amino acid changes at the interface.

[Example 11] The structure of hVB22B u2-wz4 sc (Fv) 2 is different Structure separation, structure decision

11-1. Production of humanized anti-human Mpl antibody hVB22B u2-wz4 sc (Fv) 2

The method for preparing humanized anti-Mpl antibody hVB22B u2-wz4 sc (Fv) 2 (hereinafter u2-wz4) is shown in WO2005 / 56604. This gene line uses a base sequence encoded as a linker sequence (GlyGlyGlyGlySer) x3, so that it has a base sequence consisting of VH-linker sequence-VL-linker sequence-VH-linker sequence-VL (see sequence number : 12; WO2005 / 56604 sequence number: 286). After confirming the base sequence of the gene, the DNA fragment was cloned into the expression vector pCXND3 to construct a expression vector, and gene expression was introduced into CHO-DG44 cells to produce stable expression cell lines. Specifically, a mixture of expression vector (20 μg) and 0.75 mL of CHO-DG44 cells (1 × 10 ⁷ cells / mL) suspended in PBS was cooled on ice for 10 minutes, transferred to a small glass tube, and then Gene Pulser Xcell was used. (BioRad) A pulse of 1.5 kV and a capacitance of 25 μFD was applied. After a recovery period of 10 minutes at room temperature, the electroporated cells were added to CHO-S-SFMII medium (Invitrogen) containing 500 μg / mL Geneticin (Invitrogen) and selected to establish a u2-wz4-producing CHO cell line.

The humanized antibody hVB22B u2-wz4 sc (Fv) 2 does not have a flag added, so purification from the culture supernatant uses the epitope MG10 (Gln213 to Ala231 of the human Mpl amino acid sequence) and GST Fusion protein. MG10 and GST fusion proteins were purified using glutathione Sepharose 4B (manufactured by Amersham Biosciences), and were purified according to the manufacturer's instructions. Furthermore, the purified MG10 and GST fusion proteins were fixed to HiTrap NHS-activated HP (Amersham) according to the manufacturer's instructions. (Manufactured by Biosciences) to produce affinity columns. The humanized antibody hVB22B u2-wz4 sc (Fv) 2 expresses the culture supernatant of CHO cells and flows through the MG10-GST fusion protein immobilization column, and the humanized antibody hVB22B u2-wz4 sc (Fv) 2 is adsorbed, and the humanized antibody is adsorbed at 100 mM glycan. Amino acid-HCl (pH 3.5), 0.01% Tween80 was dissolved. The dissolution fraction was immediately neutralized with 1 M Tris-HCl (pH 7.4), and gel filtration chromatography was performed using HiLoad 16/60 Superdex 200 pg (manufactured by Amersham Biosciences) to purify the monomer. The buffer of gel filtration chromatography used 20 mM citric acid buffer (pH 7.5), 300 mM NaCl, 0.01% Tween 80.

2-2. Isolation and purification of structural isomers of hVB22B u2-wz4 sc (Fv) 2

Since hVB22B u2-wz4 sc (Fv) 2 having VH ₁ - linker -VL ₂ - linker -VH ₃ - -VL sc linker sequences of ₄ (Fv) 2, and by VB22B sc (Fv) 2 Similarly, the combination of Fv (molecules that are non-covalent bonds between VH and VL) can be considered to exist as VH ₁ and VL ₂ , VH ₃ and VL ₄ each forming a bivalent scFv type of Fv, and VH ₁ and VL ₄ , VH ₂ and VL ₃ each form a single-chain bifunctional antibody-type two structural isomers of Fv (FIG. 12).

The separation of structural and structural isomers of hVB22B u2-wz4 sc (Fv) 2 was discussed. The results suggest that the use of cation exchange chromatography BioAssist S (TOSOH) can be used to separate hVB22B u2-wz4 sc (Fv) 2 Various ingredients are separated.

Mobile phase A: 20 mM sodium phosphate, pH 7.5

Mobile phase B: 20mM sodium phosphate, 500mM NaCl, pH 7.5

Flow rate: 0.8ml / min

Gradient: B 0% → B 35% (30min)

Under the above conditions, hVB22B u2-wz4 sc (Fv) 2 was separated into two peaks. The chromatogram shown in FIG. 13 is obtained, and the peaks with a short residence time are named peak1 and peak2 in this order.

For peak1 and peak2, molecular weights were measured using a Q-TOF type mass analyzer (Q Tof Ultima, Micro Mass). Q-TOF introduced the sample solution by infusion. The obtained polyvalent ion spectrum (+) was subjected to image enhancement processing (Deconvolution) using the attached software (MassLynx). . It can be seen that peak1 and peak2 have the same molecular weight.

Peptide mapping was performed on peak1 and peak2. After reduction denaturation and carboxymethylation, trypsin was used to decompose the peptide fragments, and peptide maps were obtained by reverse phase chromatography (YMC-Pack-ODS). Comparing the peptide maps of peak1 and peak2, since the patterns of the maps of peak1 and peak2 shown in FIG. 14 are the same, it can be known that the primary amino acid structures are the same.

hVB22B u2-wz4 sc (Fv) 2 has no sugar chain addition, and the molecular weights of peak1 and peak2 measured by TOF-MASS are the same, and the map patterns of peak1 and peak2 are the same. Therefore, it can be known that peak1 and peak2 have mutually different stereograms. Structural isomer.

hVB22B u2-wz4 sc (Fv) 2 Since having VH ₁ - linker -VL ₂ - linker -VH ₃ - linker sequences -VL ₄ sc (Fv) 2, as shown, configured by Fv 12 (VH and VL are non-covalently bonded molecules), there are bivalent scFv types of VH ₁ and VL ₂ , VH ₃ and VL ₄ each forming Fv, and VH ₁ and VL ₄ , VH ₂ and VL ₃ Each of the two structural isomers of the single-chain bifunctional antibody type that forms Fv, peak1 and peak2 can be considered to be one of the structures of the bivalent scFv type and the single-chain bifunctional antibody type.

As for the analysis method for identifying two structural isomers, a protease-limited decomposition method was found. The linker portion of sc (Fv) 2 is considered to have a low resistance to proteases because it has a relatively free structure. Using a protease subtilisin A, peak1 and peak2 and hVB22B u2-wz4 sc (Fv) 2 (peak1 : Peak2 ~ 1: 4) response.

20mM sodium citrate, 150mM NaCl, pH 7.5

hVB22B u2-wz4 sc (Fv) 2 peak1 or peak2: 0.15mg / mL

Subtilisin A: 10 μg / mL

37 ℃, 30min

After the reaction, Phastgel Homogeneous 12.5% was used for reduction SDS-PAGE. As a result, as shown in FIG. 15, hVB22B u2-wz4 sc (Fv) 2 bulk, peak1, and peak2 all showed the same band pattern. It can be known that the three-position linker of hVB22B u2-wz4 sc (Fv) 2 is cut to obtain the specific band of each fragment. Therefore, by using the above reaction conditions, hVB22B u2-wz4 sc (Fv ) The partial and limited decomposition of the linker part of 2.

In the structure of the bivalent scFv type and the single chain bifunctional antibody type, when one of the three linker positions is cut off, as shown in FIG. 16, in the undenatured state, it is not between VH and VL. In the covalently bonded single-chain bifunctional antibody-type structure, even if one of the three linkers is broken, the molecular weight does not change, but in the bivalent scFv type, if the central linker is broken Will produce half molecular weight molecular species. Therefore, the linker was partially cut off with the above reaction conditions, and gel filtration chromatography was performed with hVB22B u2-wz4 sc (Fv) 2 bulk, peak1, and peak2 using TSK SuperSW2000 (TOSOH). analysis. Gel filtration chromatography was performed under the following conditions.

Mobile phase: DPBS (-) pH7.4

Flow rate: 0.2ml / min

As a result, as shown in FIG. 17, it is confirmed that there is no peak portion of low molecular weight in peak2, whereas a peak portion of low molecular weight (about half molecular weight) is confirmed in peak1. The mixture hVB22B u2-wz4 sc (Fv) 2 of peak1 and peak2 was confirmed to have low molecular weight peaks in an amount corresponding to the proportion of peak1. Therefore, it is identified from this result that peak1 is a bivalent scFv type and peak2 is a single chain bifunctional antibody type.

[Example 12] Preparation of VH / VL interface variant sc (Fv) 2, analysis and identification of structural isomers

12-1. Production of VH / VL interface variant sc (Fv) 2

The assembly control using changing VH / VL interface was applied to low-molecular-weight antibody sc (Fv) 2. In order to confirm whether the formation of sc (Fv) 2 structural isomers could be controlled, a VH / VL interface variant sc ( Fv) 2.

The 39th position of the amino acid VH of u2-wz4 forming the VH / VL interface (refer to the 39th position in the amino acid sequence of 13; the sequence number of WO2005 / 56604: 289) (Refer to SEQ ID NO: 43 in the amino acid sequence of 14; WO2005 / 56604 SEQ ID: 289) Gln was changed as follows. First, make Gln (gene codon CAG) at position 39 of VH1 to Glu (gene codon GAG), Gln (gene codon CAG) at position 38 of VL2 to Glu (gene codon GAG) and VH3 39 Gln (gene codon CAG) to Lys (gene codon AAG), VL4 38 Gln (gene codon CAG) to Lys (gene codon AAG) gene hVB22B u2-wz4 (v1) sc (Fv) 2 (the following v1, the base sequence is represented by the sequence number: 15, and the amino acid sequence encoded by the base sequence is represented by the sequence number: 16). Furthermore, the production was: Gln (gene codon CAG) at position 39 of VH1 was changed to Glu (gene codon GAG), and Gln (gene codon CAG) at position 38 of VL2 was changed to Lys (gene codon AAG) and VH3 The 39th Gln (gene codon CAG) was changed to Lys (gene codon AAG), the VL4 38th Gln (gene codon CAG) was changed to Glu (gene codon GAG) gene hVB22B u2-wz4 (v3 ) sc (Fv) 2 (hereinafter, v3, the base sequence is represented by the sequence number: 17, and the amino acid sequence encoded by the base sequence is represented by the sequence number: 18). For genetic changes, QuikChange point mutation kits (manufactured by STRATAGENE) were used to introduce point mutations according to the manufacturer's instructions. After confirming the base sequence of each gene, a DNA fragment was cloned into the expression vector pCXND3 to construct a expression vector, and a stable expression cell line was produced by introducing genes into CHO-DG44 cells. The v1-producing CHO cell line and v3-producing CHO cell line were established by the method shown in Example 11.

The variants v1 and v3 were purified by the method shown in Example 11 using a MG10-GST fusion protein-immobilized column. From the results of the gel filtration chromatography shown in FIG. 18, it can be seen that the mutants v1 and v3 have less aggregates than the dimer in the culture supernatant, and the monomer ratio is compared with 59% of u2-wz4 before the change. v1 was 89% and v3 was 77%. It is speculated that the variants v1 and v3 promote the better assembly by inhibiting improper assembly due to charge repulsion due to the amino acid change at the VH / VL interface. From the above, the present assembly control successfully and efficiently represents monomer molecules.

12-2. Analysis and identification of structural isomers of VH / VL interface variant sc (Fv) 2

The existence ratios of the structural isomers of the obtained VH / VL interface variants v1, v3, and the non-variant u2-wz4 were analyzed by cation exchange chromatography and isoelectric point electrophoresis. In addition, structural identification was performed by a protease-limited decomposition method.

Cation exchange chromatography was performed as follows.

Column: TSK-gel Bioassist S, 4.6mmψ × 50mm (manufactured by TOSOH)

Flow rate: 0.8mL / min

Detection wavelength: 220nm

Dissolution conditions:

Dissolution A: 20mmol / L phosphate buffer (pH 7.0)

Eluent B: 20mmol / L phosphate buffer / 500mmol / L NaCl (pH 7.0)

gradient:

Isoelectric point electrophoresis was performed as follows. PhastGel dry IEF gel (manufactured by Amersham Biosciences) was swollen with the following gel swelling liquid for 30 minutes or more. The sample was added to the pre-swelled gel and electrophoresed using PhastSystem under the following electrophoretic conditions. After electrophoresis, soak in a 20% TCA solution for 30 minutes, and then wash with miliQ water for 5 minutes × 3 times, and perform Coomassie staining or silver staining according to the protein concentration of the sample. Coomassie's stain was stained with 0.02% CBB containing 0.1% CuSO ₄ (w / v) and decolorized with 30% methanol containing 10% acetic acid. For silver staining, a silver staining kit was used, and proteins (manufactured by Amersham Biosciences) were used, and staining was performed using the standard method of use attached to the kit.

<Gel swelling liquid>

<Electrophoresis program>

The structural identification by the protease-limited decomposition method was performed under the following conditions. Using subtilisin A, u2-wz4 refined peak1 and u2-wz4 refined peak2, and variant v1 and variant v3 were reacted under the following conditions.

20mM sodium citrate, 150mM NaCl, pH 7.5

hVB22B u2-wz4 sc (Fv) 2 peak1 or peak2: 0.15mg / mL

Subtilisin A: 10 μg / mL

37 ℃, 30min

The obtained reaction solution was analyzed by gel filtration chromatography under the following conditions.

Column: TSKgel Super2000sw (TOSOH)

Dissolution: 50mM sodium phosphate, 300mM KCl, pH 7.0

Flow rate: 0.2ml / min

Detection: 220nm

From the analysis results of the structural isomers of cation exchange chromatography and isoelectric point electrophoresis shown in Fig. 19 and Fig. 20, it can be known that u2-wz4 uses two types of 24% bivalent scFv type and 76% single chain bifunctional antibody type. The method of constructing a mixture of isomers is shown. In contrast, the variant v1 is a method of 100% single-chain bifunctional antibody-type structure isomers. The variant v3 is a 100% of a bivalent scFv-type structure isomers. Way performance. As shown in Figure 21, the results of the protease-limited decomposition also showed that the same low-molecular peaks as those of u2-wz4 refined peak1 can be observed in variant v3, and the same low-molecular peaks as that of u2-wz4 refined peak2 can be observed in variant v3. The peak. Therefore, variant v1 is expressed as a single-chain bifunctional antibody-type structural isomer, and variant v3 is expressed as a bivalent scFv-type structural isomer.

[Example 13] VH / VL interface variant sc (Fv) 2 activity evaluation and stability evaluation

13-1. Evaluation of biological activity of VH / VL interface variant sc (Fv) 2

The anti-human Mpl antibody VB22B sc (Fv) 2 was reported in the literature (Blood 2005; 105: 562-566) to exhibit TPO-like synergistic activity. Therefore, TPO-like synergistic activity using structural isoforms isolated using BaF3-human Mpl or BaF3-monkey Mpl showing TPO-dependent proliferation was evaluated.

After of RPMI1640 (Invitrogen) cells each containing 1% fetal bovine serum (Invitrogen) of washed twice, resuspended in fetal calf serum containing 10% of of RPMI1640, so that became 4 x 10 ⁵ cells / mL, and at 60μL / well min Note on 96-well plate. The concentration of rhTPO (R & D) or structural isomer samples was distributed, 40 μL was added to each well, and cultured at 37 ° C and 5% CO ₂ for 24 hours. WST-8 reagent (cell counting reagent SF, Nacalai Tesque) was added at 10 μL / well, and then the absorbance at 450 nm (control 655 nm) was measured using Benchmark Plus. After 2 hours of incubation, the absorbance at 450 nm (control 655 nm) was measured again. Since the WST-8 reagent corresponds to a color reaction of 450 nm in the number of living cells, the change in absorbance at 2 hours was used as an indicator to evaluate the similar synergistic activity of TPO.

Using purified structural isomers of VB22B sc (Fv) 2, the similar synergistic activities of TPO among BaF3-human Mpl and BaF3-monkey Mpl were evaluated, and the results are shown in FIG. 17. Comparing the synergistic activities of the structural isomers of peak1 and peak2, it can be seen that peak2 shows significantly higher activity. This suggests that the anti-Mpl antibody sc (Fv) 2 must adopt the structure of a single chain bifunctional antibody in order to exert similar synergistic activity of TPO.

The synergistic activity of VH / VL interface variants v1 and v3 was evaluated according to the method shown in Example 1. The synergistic activity varies greatly among structural isomers. As shown in FIG. 12, the peak2 of the single-chain bifunctional antibody structure shows a very high synergistic activity. In contrast, the activity of the peak1 of the bivalent scFv structure is extremely low. As shown in FIG. 22, the variants v1 and peak2 showed equivalent activity, and the variants v3 and peak1 showed approximately the same activity. From the above, it was confirmed that among the biological activities, the variant v1 forms a single-chain bifunctional antibody structure and the variant v3 forms a bivalent scFv structure.

13-2. Stability Evaluation of VH / VL Interface Variant sc (Fv) 2

Refine peak1 for u2-wz4 and refine peak2 for u2-wz4 and variant v1 For evaluation of the stability of the variant v3, a denaturation intermediate temperature (Tm value) was measured using a differential scanning calorimetry under the following conditions.

DSC: N-DSCII (Applied Thermodynamics)

Solution conditions: 20 mM sodium citrate, 300 mM NaCl, pH 7.0

Protein concentration: 0.1mg / mL

Scanning speed: 1 ° C / min

The results of each DSC measurement are shown in FIG. 23. It can be known that the Tm values of u2-wz4 refined peak2 and the mutant v1 are approximately the same as those of the non-mutant, and the stability is the same. u2-wz4 refined peak1 and variant v3 showed that the stability of variant v3 was slightly lower. In the interface control using the knobs-into-hole technology, it was reported that, for example, in the different assembly of the CH3 functional region of IgG, the Tm value of the CH3 functional region was not changed to 80.4 ° C. In contrast, the CH3 function was changed. The Tm value of the zone is 69.4 ° C, the Tm value is greatly reduced and the stability is reduced (Acta Pharmacol Sin. 2005 26 (6): 649-58). In contrast, it was confirmed that the present invention can control the assembly without reducing the stability.

Next, regarding the stability evaluation of u2-wz4 refined peak1 and u2-wz4 refined peak2 and VH / VL interface variant mutants v1 and v3, the stability evaluation by thermal acceleration test was performed under the following conditions.

<Thermal acceleration conditions>

Solution conditions: 20mM sodium citrate, pH 6.0

Protein concentration: 0.25mg / mL

Acceleration conditions: 40 ℃ -6day, 12day

Thermally accelerated samples were analyzed by gel filtration chromatography and cation exchange chromatography. The following conditions are analyzed.

As shown in FIG. 24, the results of gel filtration chromatography analysis confirmed that the residual monomer ratios of the u2-wz4 refined peak2 and the variant v1 were approximately the same, and the stability of assembly was approximately the same. In addition, the residual monomer ratios of u2-wz4 refined peak1 and variant v3 are also approximately the same, and it can be seen that the stability of assembly among the two structural isomers is approximately the same.

As shown in Figure 25, the results of cation exchange chromatography analysis show that the refined peak1 of the non-variant isomerizes to peak2 due to the isomerization reaction, and the refined peak2 of the unmutated isomerized to the isomerization reaction due to the isomerization reaction. Peak1, in contrast, the VH / VL interface variants v1 and v3 do not undergo isomerization after thermal acceleration. By applying the change of VH / VL interface, besides being able to show only one of the two structural isomers in a state of 100%, the obtained structural isomers can be stored stably without isomerization reaction. .

In this example, it was found that by using the VH / VL interface changes applied to v1 and v3, only one of the two structural isomers can be represented in a state where 100% exists. For the purpose of controlling the VH / VL interface of a single-chain antibody constructed for the purpose, it is known to use a knob-into-hole technique to control a bispecific antibody (Protein Sci. 1997 Apr 6 (4): 781-8, Remodeling domain interfaces to enhance heterodimer formation., Zhu Z, Presta LG, Zapata G, Carter P.). This method is reported to increase the formation rate of the target heterodimer structure to 72% to 92% by changing the amino acids at the total 4 positions of the VH / VL interface. In contrast, the present invention succeeded in obtaining 100 by reducing the thermal stability and the stability of structural isomers by changing the 4-position amino acid. Structure for the purpose of% ratio.

[Example 14] Humanization of a bispecific antibody with a mixed L chain

About the bispecific antibody composed of a combination of an anti-FactorIXa antibody A69-VH, an anti-FactorX antibody B26-VH, and a mixed L chain (BBA), which has the highest effect of shortening blood clotting time, humans are implemented as follows: Into.

14-1. Identical Search of Human Antibodies

Obtain human antibody amino acids from the generally published Kabat database (ftp: //ftp.ebi.ac.uk/pub/databases/kabat/) and IMGT database (http://imgt.cines.fr/) Sequence data and use the constructed database to divide into mouse A69-H chain variable regions (amino acid sequence: sequence number: 57), mouse B26-H chain variable regions (amino acid sequence: sequence number: 58 ). The mouse BBA-L chain variable region (amino acid sequence: sequence number: 59) was searched for identity. As a result, it was confirmed that they have a high degree of identity with the human antibody sequence shown below, so they were used in the framework region of humanized antibodies (hereinafter referred to as FR).

(1) A69-H chain variable region: KABATID-000064 (Kabat Database)

(Kipps et al., J Clin Invest. 1991; 87: 2087-2096)

(2) B26-H chain variable region: EMBL Accession No. AB063872 (IMGT Database)

(Unpublished data)

(3) Variable region of BBA-L chain: KABATID-024300 (Kabat Database)

(Welschof et al., J Immunol Method. 1995; 179: 203-214)

A humanized antibody in which the complementary epitope (hereinafter, CDR) of each mouse antibody was transplanted into the human antibody FR of (1) to (3) was prepared.

In addition, we use the same search sites that are generally published by NCBI. (http://www.ncbi.nlm.nih.gov/BLAST/), search for human antibody secretion signal sequences with high identity to (1)-(3) human antibodies. The secretion signal sequence shown below was obtained using the search.

(1) A69-H chain variable region: GenBank Accession No. AF062257

(2) B26-H chain variable region: GenBank Accession No. AAC18248

(3) BBA-L chain variable region: GenBank Accession No. AAA59100

14-2. Construction of humanized antibody gene expression vector

From the base sequence encoding the amino acid sequence encoding the autocrine signal sequence to the variable region of the antibody, twelve synthetic oligo DNAs of about 50 bases which are alternately bonded at about 20 bases at the 3 'end are made. Furthermore, a primer that is adhered to the 5 'end of the antibody variable region gene and has an XhoI cut sequence, and a primer that is adhered to the 3' end of the antibody variable region gene and has a SfiI cut sequence and is encoded as an intron sequence 5 'terminal sequence primer.

The synthetic oligo DNA prepared into 2.5 μM was mixed with 1 μL each, and 1 × TaKaRa Ex Taq buffer, 0.4 mM dNTPs, 0.5 units of TaKaRa Ex Taq (both manufactured by Takara Shuzo) were added to prepare 48 μL of the reaction solution. After 5 minutes of incubation at 94 ° C, a reaction consisting of 94 ° C for 2 minutes, 55 ° C for 2 minutes, and 72 ° C for 2 minutes was performed for 2 rounds, and a combination of each synthetic oligo DNA and an elongation reaction were performed. Next, 1 μL of primers (10 μM each) adhered to the 5 ′ end and the 3 ′ end of the antibody gene were added, and a reaction consisting of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute was performed for 35 rounds, and the reaction was performed at 72 ° C for 5 minutes. Minutes to amplify the antibody variable region genes. The entire amount of the reaction solution was subjected to 1% agar gel electrophoresis. The amplified fragment of the target size (about 400bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached manual, and 30 μl of sterilized water was used. Dissolve. This fragment was cloned using pGEM-T Easy Vector Systems (Promega) according to the method described in the attached instruction sheet. The base sequence of each DNA fragment was sequenced using a BigDye Terminator cycle sequencing kit (Applied Biosystems) and a DNA sequencer ABI PRISM 3730xL DNA Sequencer (Applied Biosystems) according to the method described in the attached specification.

The H chain variable region fragment confirmed to be the correct humanized antibody variable region gene sequence was inserted into the plastids and digested with XhoI and SfiI. The L chain variable region fragment was inserted into the plastids and digested with EcoRI. The reaction solution was supplied to 1% agar. Gel electrophoresis. A DNA fragment of the target size (about 400 bp) was purified by a QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and was dissolved in 30 μl of sterilized water. Then, an expression vector for animal cells was prepared as follows. In order to preferentially express the H chain as a heterocombined IgG4, the Knobs-into-hole technology of IgG1 (Non-Patent Document 3) is used to substitute an amino acid substitute for the CH3 portion of IgG4. Furthermore, in order to promote the formation of the dimer of the H chain, an amino acid substitution (-ppcpScp- → -ppcpPcp-) was also introduced into the hinge. Humanized A69 H was inserted into a pCAGGS (Niwa et al. 1991 Gene, 108: 193-199.) Expression gene with a constant region of Y349C and T366W inserted into pCAGGS (Niwa et al. 1991 Gene, 108: 193-199.). Chain variable region antibody gene fragment to make a humanized A69H chain expression vector. A humanized B26 H chain variable region antibody gene fragment was inserted into a pCAGGS expression vector in which the genes of the invariant regions of E356C, T366S, L368A, and Y407V were substituted, and a humanized B26H chain expression vector was produced. In addition, a plastid (pCAG-gκDNA) in which the wild-type antibody L chain invariant region was inserted into pCAGGS was digested with EcoRI to prepare a expression vector into which a humanized BBA L chain variable region antibody gene fragment was inserted. Ligation reaction using Rapid DNA Ligation kit (Roche Diagnostics), and the E. coli DH5α strain (Toyobo) was transformed.

14-3. Modulation of humanized bispecific antibodies

The expression of humanized bispecific antibodies was performed using the method described in Example 4-2 or the following method. HEK293H strain (Invitrogen) derived from human fetal kidney cancer cells was suspended in DMEM medium (Invitrogen) containing 10% FCS (Invitrogen), and 10 mL each was seeded at a cell density of 5 to 6 × 10 ⁵ cells / mL to a petri dish for adherent cells (10 cm in diameter, CORNING), each dish was cultured in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for one day and night, and then the medium was aspirated, and 6.9 mL of CHO-S-SFM-II (Invitrogen) medium was added. Mix 20.7 μL of 1-2 μg / mL Polyethylenimine (Polysciences Inc.) with 1-2 μL of plastid DNA mixed solution (total 13.8 μg) and mix with 690 μL of CHO-S-SFMII medium. After placement, the cells in each dish were placed in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 4 to 5 hours. Thereafter, 6.9 mL of CHO-S-SFM-II medium was added and cultured in a CO ₂ incubator for 3 days. After the culture supernatant was recovered, the cells were removed by centrifugation (about 2000 g, 5 minutes, room temperature), and passed through a 0.22 μm filter MILLEX ^(R) -GV (Millipore) for sterilization. The samples were stored at 4 ° C until use.

Next, antibody purification was performed by the method shown in Example 4-4, and antibody concentration was quantified by the method shown in Example 4-5 or the method shown below. Using BIAcore3000 (BIACORE), ProteinA was fixed to the sensor chip CM5 (BIACORE). Specifically, according to the manufacturer's instructions, the activated sensor chip was reacted with a protein A solution diluted to 50 μg / mL with a 10 mM sodium acetate aqueous solution (pH 4.0, BIACORE) at 5 μL / min for 30 minutes. A blocking operation is performed to make a ProteinA immobilized sensor chip. Using this sensor chip, the concentration of the culture supernatant and refined product was measured using BIAcore Q. HBS-EP buffer solution (BIACORE) was used for the sensor chip fixing and concentration measurement. In addition, as a standard for concentration measurement, human IgG4 (human-type anti-TF antibody, refer to WO 99/51743) was used, which was diluted in a 2-fold series in 6 stages from 2000 ng / mL in HBS-EP buffer.

14-4. Evaluation of the activity of humanized bispecific antibodies and changes in antibody sequences

In order to evaluate the plasma coagulation ability of the prepared humanized bispecific antibodies and chimeric bispecific antibodies (A69 / B26 / BBA), the effect of antibodies on APTT was investigated using F.VIII-deficient plasma according to the method of Example 5. For humanized bispecific antibodies with low blood coagulation ability, the amino acid of human antibody FR is changed with the aim of increasing activity. Furthermore, when expressing secretion, three types of antibodies, namely humanized A69 / humanized BBA antibody, humanized B26 / humanized BBA antibody, humanized A69 / humanized B26 / humanized BBA, were expressed. Isolation of the antibody, only purification of the bispecific antibody, the amino acid change that decreases the isoelectric point of the humanized A69 H chain variable region, and the rise of the isoelectric point of the humanized B26 H chain variable region. Specifically, a QuikChange point mutation kit (Stratagene) was used to introduce mutations into the variable region of a humanized antibody by the method described in the attached specification. The H chain variable region fragment of the humanized antibody variable region gene sequence identified as the target was inserted into the plastids and digested with XhoI and SfiI, and the L chain variable region fragment was inserted into the plastids and digested with EcoRI. The reaction solution was subjected to 1% agar coagulation. Gel electrophoresis. A DNA fragment of the desired size (about 400 bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction manual and dissolved in 30 μl of sterilized water. Out. Thereafter, the expression vector for animal cells was prepared by the method shown in Example 14-2. The humanized bispecific antibody was prepared by the method shown in Example 14-3, and the blood coagulation activity was evaluated by the method shown in Example 5.

Evaluation of the amino acid changes and blood clotting ability of the repeated FR sequence, and a humanized bispecific antibody (humanized A69 (hA69-PFL) /) with the same activity as the chimeric bispecific antibody (A69 / B26 / BBA) was obtained. Humanized B26 (hB26-PF) / Humanized BBA (hAL-AQ)) (Figure 26). The sequence of the variable region of each antibody is shown in the following sequence numbers.

(1) Humanized A69 antibody VH (hA69-PFL) sequence number: 19 (base sequence), sequence number: 20 (amino acid sequence)

(2) Humanized B26 antibody VH (hB26-PF) sequence number: 21 (base sequence), sequence number: 22 (amino acid sequence)

(3) Humanized BBA antibody VL (hAL-AQ) sequence number: 23 (base sequence), sequence number: 24 (amino acid sequence)

[Example 15] Amino acid change positions were selected in order to improve the formation efficiency of the bispecific antibody in the upward-constant region

The aim is to change the amino acid existing at the CH3 interface in the constant region and use charge repulsion to increase the formation efficiency of heterodimeric bispecific antibodies. First, using the crystal structure of the CH3 region (Protein Data bank, PDB code 1OQX), the paired amino acids that form electrostatic interactions when CH3 and dimers are formed are explored. As a result, it was found that in the interface when the CH3 homodimer was formed, three pairs of H chains 356 and 439, 357 and 370, 399 and 409 (numbered as EU number (Kabat EA et al. 1991) .Sequences of Proteins of Immunological Interest.NIH)) have positive and negative charges and have static electricity Interact and choose to change position. The method of change is considered to promote the formation of heterodimers by substituting and changing the charge of the positive charge and the negative charge amino acid that become a pair. This control principle is shown in Figure 27. In addition, I also tried to introduce changes in the disulfide bond at the CH3 interface. The modified amino acid positions are summarized in Table 1.

[Example 16] Amino acid changes at the CH3 interface of the invariant region of a humanized bispecific antibody

In order to change the amino acid at the CH3 interface of the H chain invariant region selected in Example 15, the following operations were performed. The base sequence of the two amino acids (Ala-Ser) encoding the N-terminal side of the H-chain invariant region using the genes of the H-chain invariant region of human IgG1 and human IgG4 as templates is designed to be a NheI recognition sequence (GCTAGC) The 5 'end primers are glued at the 3' end, and each H chain invariant region is amplified by PCR using a primer designed with a NotI recognition site, and the pBluescriptKS + vector (Toyobo) is made with NheI, NotI (both PBCH (containing IgG1 invariant region genes) and pBCH4 (containing IgG4 invariant region genes) digested by vector. Use primers that are complementary to the 5 'terminal base sequence of the H chain variable region of the humanized A69 antibody and humanized B26 antibody and have the Zacquick sequence (CCACC) and EcoRI recognition sequence and the 3' end with the NheI recognition sequence The base sequence was subjected to PCR using primers, and the obtained PCR product was digested with EcoRI and NheI (both made by Takara Shuji), inserted into pBCH or pBCH4, and linked to the variable region and the constant region. Next, in order to change the amino acid present at the CH3 interface of the H chain invariant region, a mutation was introduced into the H chain invariant region using the QuikChange point mutation kit (Stratagene) by the method described in the attached specification. The H chain gene fragment identified as the gene sequence of the H chain invariant region of interest was inserted into plastids and digested with EcoRI and NotI (both made by Takara Shuji), and the reaction solution was supplied with 1% agar Gel electrophoresis. The H-chain gene fragment of the target size (about 1400 bp) was purified using the QIAquick Gel Extraction Kit (QIAGEN) according to the method described in the attached instruction manual, and dissolved in 30 μl of sterilized water. Then, pCAGGS digested with EcoRI and NotI was inserted, and expression plastids were produced. The preparation of humanized bispecific antibodies was performed according to the method shown in Example 14-3. The modified amino acid positions are summarized in Table 1. The change position number in Table 1 uses the EU number (Kabat EA et al. 1991. Sequences of Proteins of Immunological Interest. NIH). The letter before changing the position number is the single letter code of the amino acid before the change, and the letter after changing the position number is the single letter code of the amino acid after the change.

In the above table, KiH indicates a variant using the Knobs-into-holes technique of Non-Patent Document 3.

[Example 17] Evaluation of formation efficiency and stability of a bispecific antibody (IgG4 type) with a changed CH3 interface

About IgG4 wild type, KiH, s1, s2, s3, w1, w2 w3, s1C, s2C, s3C, w3C, and w3C2 were analyzed by cation exchange chromatography (IEX) to evaluate the formation efficiency of bispecific antibodies (hereinafter referred to as BiAb). The cation exchange chromatography analysis conditions are as follows. The homodimer A-Homo of humanized A69 antibody, the heterodimer BiAb of humanized A69 antibody and humanized B26 antibody, and the homodimer B-Homo of humanized B26 antibody were calculated. Peak area ratio.

Column: ProPac WCX-10, 4 × 250mm, (Dionex)

Mobile phase: A: 10mmol / L NaH ₂ PO ₄ / Na ₂ HPO ₄ , pH 6.25

B: 10mmol / L NaH ₂ PO ₄ / Na ₂ HPO ₄ , 500mmol / L NaCl, pH 6.25

Flow rate: 1.0mL / min

Gradient: 10% B (5min) → (40min) → 60% B → (5min) → 100% B (5min)

Detection: 220nm

Regarding wild type, KiH, s2, s3, s1C, s2C, s3C, w3C, and w3C2, the BiAb peaks were fractionated during the IEX analysis, and BiAb was purified. The BiAb fractions were concentrated with Amicon Ultra, MWCO 10000 (Millipore), and dialysis was performed overnight in a cold place with 20 mM sodium acetate, 150 mM NaCl, pH 6.0, and then recovered, and the concentration of BiAb was unified to 0.1 mg / mL. Two vials were used as the starting point, and the stability test was performed at 60 ° C for 1 week and at 60 ° C for 1 week. The analysis was performed by gel filtration chromatography (SEC), and the monomer peak residual ratio was calculated (the area of the monomer peak of the sample at 60 ° C. for 1 week / the area of the monomer peak of the starting sample × 100). The gel filtration chromatography analysis conditions are as follows.

Column: Super3000 (TOSOH)

Mobile phase: 50mM sodium phosphate, 300mM KCl, pH 7.0

Flow rate: 0.2ml / min

Detection: 220nm

The IEX chromatograms of IgG4 wild type, s1, s2, s3, and w1 are shown in Figure 28. The formation ratios of -Homo, BiAb, and B-Homo are shown in Figure 29. In addition, the residual monomer ratio after 60 ° C for 1 week is shown in Fig. 30.

The CH3 interface variants found in this example are shown in Figs. 28 and 29. Compared with the wild type, the target BiAb formation efficiency is greatly improved. Since CH3 is located in the invariant region, since the natural amino acid is changed, from the viewpoint of antigenicity, it is desirable to change the position less. KiH applies a total of 6 position changes: used to introduce a total of 4 positions of the two H-chain meters, and disulfide bond introduction 2 positions. Therefore, as shown in FIG. 29, a high BiAb formation efficiency can be obtained. However, from the results of the stability test shown in FIG. 30, the thermal stability was significantly decreased in comparison with the wild type even though a disulfide bond was introduced. In order to develop antibodies into medical drugs, a sizing agent is required, and therefore high thermal stability is desired.

On the other hand, compared with the wild-type CH3 interface variants found in this example, the target BiAb formation efficiency was successfully greatly improved. Among these variants, for example, s2, s3, w1, w2, w3, and s1C have less changes than KiH (6 position change). For a total of 2 or 4 position changes, a height of more than 90% can be obtained. The efficiency of BiAb formation is considered to be a small risk of antigenicity. Moreover, from the results of the stability test shown in FIG. 30, among the variants, for example, s2, s3, w3, w3C, and w3C2 have a high BiAb formation efficiency of 90% or more, and have higher thermal stability than KiH (single Body survival rate is high), and s3, s2c, s3C, w3C, and w3C2 have higher thermal stability than wild type, and are useful for developing stable pharmaceutical preparations.

In this example, it was found that by changing the amino acids in the H chain Nos. 356, 357, 370, 399, 409, 439, and 439 in the CH3 interface and introducing a molecular repulsion force generated by the charge, the Objective BiAb formation efficiency is greatly improved. In addition, through the introduction of these separate, combined, and disulfide bonds, BiAb formation efficiency can be greatly improved with fewer changes than KiH, and it has higher stability than KiH, and has higher thermal stability and BiAb formation than wild type. Significantly improved efficiency.

[Example 18] Evaluation of coagulation activity of a bispecific antibody with a modified CH3 interface

Using the purified IgG4-type bispecific antibody (s1, s2, s3, w1, w2, w3) whose CH3 interface was modified in Example 16, the coagulation activity was evaluated according to the method shown in Example 5. Since the coagulation activity does not change even if the amino acid at the CH3 interface of the constant region shown in FIG. 31 is changed, it means that the amino acid change at the CH3 interface does not affect the structure of the variable region related to the reaction of the antigen.

[Example 19] Evaluation of the formation efficiency of a bispecific antibody (IgG1 type) with a modified CH3 interface

The wild type, KiH, w1, w2, and w3 of the IgG1 type were analyzed by cation exchange chromatography (IEX) to evaluate the efficiency of BiAb formation. The cation exchange chromatography analysis conditions are as follows. The homodimer A-Homo of humanized A69 antibody, the heterodimer BiAb of humanized A69 antibody and humanized B26 antibody, and the homodimer B-Homo of humanized B26 antibody were calculated. Peak area ratio.

Column: ProPac WCX-10, 4 × 250mm, (Dionex)

Mobile phase: A: 10mmol / L NaH ₂ PO ₄ / Na ₂ HPO ₄ , pH 6.25

B: 10mmol / L NaH ₂ PO ₄ / Na ₂ HPO ₄ , 500mmol / L NaCl, pH 6.25

Flow rate: 1.0mL / min

Gradient: 10% B (5min) → (40min) → 60% B → (5min) → 100% B (5min)

Detection: 220nm

The formation ratios of IgG1 type wild type, KiH, w1, w2, w3 A-Homo, BiAb, and B-Homo are shown in FIG. 32. As with the IgG4 type, compared with the wild type, the target BiAb formation efficiency is significantly increased. Similar to the IgG4 type, it is considered that the risk of antigenicity is small by obtaining a high BiAb formation efficiency of 90% or more by changing 4 positions less than KiH. In this example, it was found that the method of changing the amino acids in the H chain 356, 357, 370, 399, 409, and 439 in the CH3 interface is not only the subtype of the constant region of the antibody is IgG4, but also Suitable for IgG1 and can be used for all IgG antibodies.

[Industrial availability]

In the method of the present invention, since the number of amino acid substitutions is small, it is possible to control the assembly without changing the structure and function (activity) of the original polypeptide, which is very useful. In addition, the effect on antigenicity is small.

By using the method of the present invention, a bispecific antibody that actually retains activity can be efficiently obtained.

<110> CHUGAI SEIYAKU KABUSHIKI KAISHA

<120> Method for preparing desired molecules comprising polypeptides by inhibiting interaction between the polypeptides

<130> C1-A0415Y1-TW

<150> JP 2005-101105

<151> 2005-03-31

<150> JP 2005-378266

<151> 2005-12-28

<160> 59

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Claims (10)

A method for manufacturing a polypeptide variant is to mutate amino acid residues existing at the interface between VH and VL to control the assembly of the polypeptide, including (a) changing the polypeptide forming two or more structural isomers to change Nucleic acid encoding an amino acid residue existing at the interface between VH and VL, which changes the original nucleic acid in a manner that inhibits the internal assembly of polypeptides that undesirably form more than one structural isomer, (b) cultivates the host The cell expresses the nucleic acid, (c) recovering the polypeptide from the host cell culture, wherein the polypeptide is a single-chain polypeptide in which two or more heavy chain variable regions and two or more light chain variable regions are bound by a linker, In addition, the change of step (a) is a method in which the original nucleic acid is replaced to change the amino acid residues of the two or more residues forming the interface to the same positive charge, and an amino acid residue is introduced into the interface. The amino acid residues that form two or more residues at the interface are at least one group of amino acid residues selected due to the group of amino acid residues on the heavy and light chain FR2 regions. Group, where the group of amino acid residues is the following (i) and (ii) The group of at least one amino acid residue selected from the group: (i) the Kabat number 39 on the variable region of the heavy chain and the Kabat number on the variable region of the light chain are 38 amino acid residues; (ii) Kabat number 45 amino acid residues on the heavy chain variable region and 44 amino acid residues Kabat number 44 on the light chain variable region, wherein the introduction The amino acid residues are selected from the group consisting of free lysine (K), arginine (R) and histidine (H). A method for manufacturing heteromultimers is to make mutations in amino acid residues forming the interface between polypeptides to control the assembly of heteromultimers, including: (a) forming heterogeneous species of two or more multimers Nucleic acid-encoding nucleic acid in a multimer that changes the amino acid residues that form the interface between polypeptides is to alter the original nucleic acid in a manner that inhibits the assembly between polypeptides that undesirably form more than one polymer, (b) Culturing a host cell to express the nucleic acid, and (c) recovering the heteromultimer from the host cell culture, wherein the heteromultimer system includes multiple specific antibodies of two or more heavy chain variable regions, and wherein The change of step (a) is a method in which the original nucleic acid is replaced to change the amino acid residues of the two or more residues forming the interface to the same kind of positive charge, which introduces a mutation of an amino acid residue into the interface. The amino acid residues with more than 2 residues forming the interface are a group of amino acid residues selected from at least one group of (1) to (3) below: (1) in the heavy chain and The group of amino acid residues on the FR2 region of the light chain is selected by the following groups (i) and (ii) Group of at least one amino acid residue: (i) amino acid residue with Kabat number 39 on the variable region of the heavy chain and amino acid residue with Kabat number 38 on the variable region of the light chain Residues; (ii) amino acid residues with Kabat number 45 on the variable region of the heavy chain and amino acid residues with Kabat number 44 on the variable region of the light chain; and (2) contained in human origin The group of amino acid residues in the IgG-type heavy chain CH3 domain is at least one group of amino acid residue groups selected from the following groups (i) to (iii): (i) is included in Amino acid residues in heavy chain CH3 region, amino acid residues with EU numbers 356 and 439; (ii) Amino acid residues included in heavy chain CH3 region, EU number with 357 and 370 amino acid residues Residues; (iii) amino acid residues included in the CH3 region of the heavy chain, amino acid residues of EU numbers 399 and 409: (3) amino acid residues of Kabat number 221 on the heavy chain CH1 region Kabat numbered 123 amino acid residues on the CL region of the light chain and light chain, wherein the introduced amino acid residues are selected from free amino acids (K), arginine (R) and histidine (H ). The method for manufacturing a heteromultimer as described in item 2 of the scope of patent application, wherein the heteromultimer is a pair of specific antibodies. A polypeptide variant that changes the amino acid residues that form the internal interface of the polypeptide to inhibit undesired formation of the original polypeptide that can form two or more structural isomers. Assembly, in which the polypeptide is a single-chain polypeptide that combines two or more heavy chain variable regions and two or more light chain variable regions with a linker, and wherein the amino acid residues forming the interface within the polypeptide are changed In order to form an amino acid residue with more than 2 residues at the interface, the amino acid residues at the interface will be replaced with the same kind of positive charge to form an amino acid residue with more than 2 residues at the interface. The group is at least one group of amino acid residues selected due to the group of amino acid residues on the FR2 region of the heavy and light chains. Here, the group of amino acid residues is given by The group of at least one amino acid residue selected from the following groups (i) and (ii): (i) the amino acid residue with a Kabat number 39 on the variable region of the heavy chain and the light chain Kabat numbered 38 amino acid residues on the variable region; (ii) Kabat numbered 45 amino acid residues on the variable region of the heavy chain and light The amino acid residue with Kabat number 44 on the variable region of the chain, the introduced amino acid residue is selected from free lysine (K), arginine (R) and histidine (H) Groups of people. A heteromultimer that changes the amino acid residues that form the interface between the polypeptides to inhibit the undesired assembly of the original polypeptide that forms one or more polymers between the original polypeptides that can form two or more polymers Wherein the heteromultimeric system includes multiple specific antibodies of two or more heavy chain variable regions, and wherein the amino acid residues forming the interface between the polypeptides are changed to form amines having more than 2 residues at the interface The amino acid residues will be replaced with amino acid residues in the interface with the same kind of positive charge to form amino acid residues with more than 2 residues in the interface. The reason is shown in (1) below. (3) the group of amino acid residues in the group of at least one group of amino acids selected: (1) the group of amino acid residues on the heavy and light chain FR2 regions, The group of at least one amino acid residue selected from the following groups (i) and (ii): (i) the amino acid residue with a Kabat number 39 on the variable region of the heavy chain and the light chain Kabat numbered 38 amino acid residues on the variable region; (ii) Kabat numbered 45 amino acid residues on the heavy chain variable region and Kabat on the light chain variable region The amino acid residue No. 44; and (2) the group of amino acid residues included in the heavy chain CH3 domain from human IgG-type, which is represented by the following groups (i) to (iii) Selected at least one group of amino acid residues: (i) amino acid residues included in the CH3 region of the heavy chain, amino acid residues of EU numbers 356 and 439; (ii) included in the heavy chain CH3 Amino acid residues in the region, EU residues of 357 and 370; (iii) Amino acid residues in the heavy chain CH3 region, EU residues of 399 and 409: (3) Amino acid residue with Kabat number 221 on the CH1 region of the heavy chain and 123 amino acid residue with Kabat number 123 on the CL region of the light chain, wherein the introduced amino acid residue is a selective free ion A group of amino acids (K), arginine (R) and histidine (H). The heteromultimer as described in item 5 of the scope of patent application, wherein the heteromultimer is a pair of specific antibodies. A composition comprises a polypeptide variant according to claim 4 or a heteromultimer according to claim 5 and a pharmaceutically acceptable carrier. A nucleic acid that encodes a polypeptide variant of claim 4 or a heteromultimer of claim 5. A host cell having a nucleic acid having the scope of patent application No. 8. A method for manufacturing a polypeptide variant as claimed in item 4 of the patent application or a heteromultimer as claimed in item 5 of the patent application, comprising: culturing host cells in the application for item 9 of the patent application, and recovering the polypeptide from the cell culture.